diff --git a/src/spatialdata_plot/pl/utils.py b/src/spatialdata_plot/pl/utils.py
index 6068533f..117ffbba 100644
--- a/src/spatialdata_plot/pl/utils.py
+++ b/src/spatialdata_plot/pl/utils.py
@@ -3194,6 +3194,69 @@ def _prepare_transformation(
     return trans, trans_data
 
 
+def _apply_cmap_alpha_to_datashader_result(
+    result: Any,
+    agg: DataArray,
+    cmap: str | list[str] | Colormap,
+    span: list[float] | tuple[float, float] | None,
+) -> Any:
+    """Apply the colormap's alpha channel to a datashader RGBA result.
+
+    Datashader ignores the per-entry alpha channel of matplotlib colormaps,
+    so pixels that the cmap marks as transparent (alpha=0) are rendered
+    opaque.  This function post-processes the shaded RGBA output to restore
+    the cmap's intended transparency.  See :issue:`376`.
+    """
+    if not isinstance(cmap, Colormap):
+        return result
+
+    # Quick check: does this cmap have any transparent entries?
+    test_vals = np.linspace(0, 1, min(cmap.N, 256))
+    cmap_alphas = cmap(test_vals)[:, 3]
+    if np.all(cmap_alphas >= 1.0):
+        return result
+
+    # Get or ensure we have an (H, W, 4) uint8 array
+    if hasattr(result, "values"):
+        # datashader Image — uint32 packed, convert via to_numpy()
+        rgba = result.to_numpy().base
+        if rgba is None:
+            return result
+    else:
+        rgba = result
+
+    if rgba.ndim != 3 or rgba.shape[2] != 4:
+        return result
+
+    # Normalise aggregate values to [0, 1] using the same span datashader used
+    agg_vals = agg.values.astype(np.float64)
+    valid = np.isfinite(agg_vals)
+    if not valid.any():
+        return result
+
+    if span is not None:
+        lo, hi = float(span[0]), float(span[1])
+    else:
+        lo = float(np.nanmin(agg_vals))
+        hi = float(np.nanmax(agg_vals))
+
+    if hi <= lo or not np.isfinite(lo) or not np.isfinite(hi):
+        return result
+
+    normed = np.clip((agg_vals - lo) / (hi - lo), 0.0, 1.0)
+
+    # Look up cmap alpha for each pixel
+    desired_alpha = cmap(normed)[:, :, 3]
+
+    # Zero out pixels where the cmap wants transparency
+    transparent = valid & (desired_alpha < 1.0)
+    if transparent.any():
+        # Scale the existing alpha by the cmap's alpha
+        rgba[transparent, 3] = (rgba[transparent, 3].astype(np.float32) * desired_alpha[transparent]).astype(np.uint8)
+
+    return result
+
+
 def _datashader_map_aggregate_to_color(
     agg: DataArray,
     cmap: str | list[str] | ListedColormap,
@@ -3245,9 +3308,10 @@ def _datashader_map_aggregate_to_color(
         img_over = img_over.to_numpy().base
         if img_over is not None:
             stack[stack[:, :, 3] == 0] = img_over[stack[:, :, 3] == 0]
-        return stack
 
-    return ds.tf.shade(
+        return _apply_cmap_alpha_to_datashader_result(stack, agg, cmap, span)
+
+    result = ds.tf.shade(
         agg,
         cmap=cmap,
         color_key=color_key,
@@ -3255,6 +3319,7 @@ def _datashader_map_aggregate_to_color(
         span=span,
         how="linear",
     )
+    return _apply_cmap_alpha_to_datashader_result(result, agg, cmap, span)
 
 
 def _hex_no_alpha(hex: str) -> str:
diff --git a/tests/_images/Utils_transparent_cmap_shapes_clip_false.png b/tests/_images/Utils_transparent_cmap_shapes_clip_false.png
new file mode 100644
index 00000000..ea15e0ad
Binary files /dev/null and b/tests/_images/Utils_transparent_cmap_shapes_clip_false.png differ
diff --git a/tests/_images/Utils_transparent_cmap_shapes_datashader.png b/tests/_images/Utils_transparent_cmap_shapes_datashader.png
new file mode 100644
index 00000000..ea15e0ad
Binary files /dev/null and b/tests/_images/Utils_transparent_cmap_shapes_datashader.png differ
diff --git a/tests/_images/Utils_transparent_cmap_shapes_matplotlib.png b/tests/_images/Utils_transparent_cmap_shapes_matplotlib.png
new file mode 100644
index 00000000..8af4cd35
Binary files /dev/null and b/tests/_images/Utils_transparent_cmap_shapes_matplotlib.png differ
diff --git a/tests/pl/test_utils.py b/tests/pl/test_utils.py
index a9296d2e..42165333 100644
--- a/tests/pl/test_utils.py
+++ b/tests/pl/test_utils.py
@@ -4,10 +4,16 @@
 import pandas as pd
 import pytest
 import scanpy as sc
+import xarray as xr
 from spatialdata import SpatialData
 
 import spatialdata_plot
-from spatialdata_plot.pl.utils import _get_subplots
+from spatialdata_plot.pl.utils import (
+    _apply_cmap_alpha_to_datashader_result,
+    _datashader_map_aggregate_to_color,
+    _get_subplots,
+    set_zero_in_cmap_to_transparent,
+)
 from tests.conftest import DPI, PlotTester, PlotTesterMeta
 
 sc.pl.set_rcParams_defaults()
@@ -52,8 +58,6 @@ def test_plot_colnames_that_are_valid_matplotlib_greyscale_colors_are_not_evalua
         sdata_blobs.pl.render_shapes("blobs_polygons", color=colname).pl.show()
 
     def test_plot_can_set_zero_in_cmap_to_transparent(self, sdata_blobs: SpatialData):
-        from spatialdata_plot.pl.utils import set_zero_in_cmap_to_transparent
-
         # set up figure and modify the data to add 0s
         _, axs = plt.subplots(nrows=1, ncols=2, layout="tight")
         sdata_blobs.tables["table"].obs["my_var"] = list(range(len(sdata_blobs.tables["table"].obs)))
@@ -73,6 +77,49 @@ def test_plot_can_set_zero_in_cmap_to_transparent(self, sdata_blobs: SpatialData
             ax=axs[1], colorbar=False
         )
 
+    def _render_transparent_cmap_shapes(self, sdata_blobs: SpatialData, method: str):
+        _, axs = plt.subplots(nrows=1, ncols=2, layout="tight")
+        new_cmap = set_zero_in_cmap_to_transparent(cmap="viridis")
+        sdata_blobs["table"].obs["region"] = pd.Categorical(["blobs_polygons"] * sdata_blobs["table"].n_obs)
+        sdata_blobs["table"].uns["spatialdata_attrs"]["region"] = "blobs_polygons"
+        sdata_blobs.shapes["blobs_polygons"]["value"] = [0.0, 2.0, 3.0, 4.0, 5.0]
+
+        # left: baseline with standard viridis
+        sdata_blobs.pl.render_images("blobs_image").pl.render_shapes(
+            "blobs_polygons", color="value", cmap="viridis", method=method
+        ).pl.show(ax=axs[0], colorbar=False)
+
+        # right: transparent cmap — shape with value=0 should reveal the image
+        sdata_blobs.pl.render_images("blobs_image").pl.render_shapes(
+            "blobs_polygons", color="value", cmap=new_cmap, method=method
+        ).pl.show(ax=axs[1], colorbar=False)
+
+    def test_plot_transparent_cmap_shapes_matplotlib(self, sdata_blobs: SpatialData):
+        self._render_transparent_cmap_shapes(sdata_blobs, method="matplotlib")
+
+    def test_plot_transparent_cmap_shapes_datashader(self, sdata_blobs: SpatialData):
+        self._render_transparent_cmap_shapes(sdata_blobs, method="datashader")
+
+    def test_plot_transparent_cmap_shapes_clip_false(self, sdata_blobs: SpatialData):
+        """Transparent cmap with clip=False norm (3-part shading path)."""
+        from matplotlib.colors import Normalize
+
+        _, axs = plt.subplots(nrows=1, ncols=2, layout="tight")
+        new_cmap = set_zero_in_cmap_to_transparent(cmap="viridis")
+        norm = Normalize(vmin=0, vmax=5, clip=False)
+
+        sdata_blobs["table"].obs["region"] = pd.Categorical(["blobs_polygons"] * sdata_blobs["table"].n_obs)
+        sdata_blobs["table"].uns["spatialdata_attrs"]["region"] = "blobs_polygons"
+        sdata_blobs.shapes["blobs_polygons"]["value"] = [0.0, 2.0, 3.0, 4.0, 5.0]
+
+        sdata_blobs.pl.render_images("blobs_image").pl.render_shapes(
+            "blobs_polygons", color="value", cmap="viridis", norm=norm, method="datashader"
+        ).pl.show(ax=axs[0], colorbar=False)
+
+        sdata_blobs.pl.render_images("blobs_image").pl.render_shapes(
+            "blobs_polygons", color="value", cmap=new_cmap, norm=norm, method="datashader"
+        ).pl.show(ax=axs[1], colorbar=False)
+
 
 @pytest.mark.parametrize(
     "color_result",
@@ -90,6 +137,79 @@ def test_is_color_like(color_result: tuple[ColorLike, bool]):
     assert spatialdata_plot.pl.utils._is_color_like(color) == result
 
 
+class TestCmapAlphaDatashader:
+    """Regression tests for #376: set_zero_in_cmap_to_transparent with datashader."""
+
+    def test_transparent_pixels_get_alpha_zero(self):
+        """Post-processing sets alpha=0 for pixels mapping to transparent cmap entries."""
+        import datashader as ds
+
+        cmap = set_zero_in_cmap_to_transparent("viridis")
+        data = np.array([[0.0, 5.0, 10.0]], dtype=np.float64)
+        agg = xr.DataArray(data, dims=["y", "x"])
+
+        shaded = ds.tf.shade(agg, cmap=cmap, min_alpha=254, how="linear")
+        result = _apply_cmap_alpha_to_datashader_result(shaded, agg, cmap, span=[0.0, 10.0])
+        rgba = result.to_numpy().base if hasattr(result, "to_numpy") else result
+
+        assert rgba[0, 0, 3] == 0, f"Expected alpha=0 at value=0.0, got {rgba[0, 0, 3]}"
+        assert rgba[0, 1, 3] > 0, "Expected non-zero alpha at value=5.0"
+        assert rgba[0, 2, 3] > 0, "Expected non-zero alpha at value=10.0"
+
+    def test_opaque_cmap_unchanged(self):
+        """Post-processing is a no-op for fully opaque cmaps."""
+        import datashader as ds
+
+        cmap = plt.get_cmap("viridis")
+        data = np.array([[0.0, 5.0, 10.0]], dtype=np.float64)
+        agg = xr.DataArray(data, dims=["y", "x"])
+
+        shaded = ds.tf.shade(agg, cmap=cmap, min_alpha=254, how="linear")
+        rgba_before = shaded.to_numpy().base.copy()
+        result = _apply_cmap_alpha_to_datashader_result(shaded, agg, cmap, span=[0.0, 10.0])
+        rgba_after = result.to_numpy().base if hasattr(result, "to_numpy") else result
+        np.testing.assert_array_equal(rgba_before, rgba_after)
+
+    def test_string_cmap_passthrough(self):
+        """Post-processing is a no-op for string cmaps (early return)."""
+        dummy_rgba = np.zeros((2, 3, 4), dtype=np.uint8)
+        dummy_rgba[:, :, 3] = 200
+        data = np.array([[0.0, 5.0, 10.0]], dtype=np.float64)
+        agg = xr.DataArray(data, dims=["y", "x"])
+
+        result = _apply_cmap_alpha_to_datashader_result(dummy_rgba, agg, "viridis", span=[0.0, 10.0])
+        np.testing.assert_array_equal(result, dummy_rgba)
+
+    def test_end_to_end_datashader_map(self):
+        """_datashader_map_aggregate_to_color produces alpha=0 for transparent cmap entries."""
+        cmap = set_zero_in_cmap_to_transparent("viridis")
+        data = np.array([[0.0, 5.0, 10.0]], dtype=np.float64)
+        agg = xr.DataArray(data, dims=["y", "x"])
+
+        result = _datashader_map_aggregate_to_color(agg, cmap=cmap, min_alpha=254, span=[0.0, 10.0])
+        img = result.to_numpy().base if hasattr(result, "to_numpy") else result
+
+        assert img[0, 0, 3] == 0, f"Expected alpha=0 at value=0.0, got {img[0, 0, 3]}"
+        assert img[0, 1, 3] > 0, "Expected non-zero alpha at value=5.0"
+
+    def test_span_none_preserves_colors(self):
+        """With span=None, non-transparent shapes keep their correct colors."""
+        cmap = set_zero_in_cmap_to_transparent("viridis")
+        data = np.array([[0.0, 5.0, 10.0]], dtype=np.float64)
+        agg = xr.DataArray(data, dims=["y", "x"])
+
+        result = _datashader_map_aggregate_to_color(agg, cmap=cmap, min_alpha=254)
+        img = result.to_numpy().base if hasattr(result, "to_numpy") else result
+
+        # value=0 should be transparent
+        assert img[0, 0, 3] == 0
+        # value=5 and value=10 should be opaque with correct viridis colors (not white)
+        assert img[0, 1, 3] > 0
+        assert img[0, 2, 3] > 0
+        # The non-transparent pixels should NOT be white (R=255,G=255,B=255)
+        assert not (img[0, 1, 0] == 255 and img[0, 1, 1] == 255 and img[0, 1, 2] == 255)
+
+
 def test_extract_scalar_value():
     """Test the new _extract_scalar_value function for robust numeric conversion."""