diff --git a/changes/4493.bugfix.md b/changes/4493.bugfix.md
new file mode 100644
index 0000000000..e4c2c708c9
--- /dev/null
+++ b/changes/4493.bugfix.md
@@ -0,0 +1 @@
+Accept a list of byte values as a ``fill_value`` for byte data types, as permitted by the Zarr V3 spec.
diff --git a/src/zarr/core/dtype/npy/bytes.py b/src/zarr/core/dtype/npy/bytes.py
index daeb105819..511db575d4 100644
--- a/src/zarr/core/dtype/npy/bytes.py
+++ b/src/zarr/core/dtype/npy/bytes.py
@@ -18,7 +18,7 @@
     check_dtype_spec_v2,
     v3_unstable_dtype_warning,
 )
-from zarr.core.dtype.npy.common import check_json_str
+from zarr.core.dtype.npy.common import check_json_byte_list, check_json_str
 from zarr.core.dtype.wrapper import TBaseDType, ZDType
 from zarr.errors import DataTypeValidationError
 
@@ -524,8 +524,10 @@ def from_json_scalar(self, data: JSON, *, zarr_format: ZarrFormat) -> np.bytes_:
 
         if check_json_str(data):
             return self.to_native_dtype().type(base64.standard_b64decode(data.encode("ascii")))
+        if check_json_byte_list(data):
+            return self.to_native_dtype().type(bytes(data))
         raise TypeError(
-            f"Invalid type: {data}. Expected a base64-encoded string."
+            f"Invalid type: {data}. Expected a base64-encoded string or a list of byte values."
         )  # pragma: no cover
 
     @property
@@ -919,7 +921,11 @@ def from_json_scalar(self, data: JSON, *, zarr_format: ZarrFormat) -> np.void:
         """
         if check_json_str(data):
             return self.to_native_dtype().type(base64.standard_b64decode(data))
-        raise TypeError(f"Invalid type: {data}. Expected a string.")  # pragma: no cover
+        if check_json_byte_list(data):
+            return self.to_native_dtype().type(bytes(data))
+        raise TypeError(
+            f"Invalid type: {data}. Expected a base64-encoded string or a list of byte values."
+        )  # pragma: no cover
 
     @property
     def item_size(self) -> int:
@@ -1200,7 +1206,11 @@ def from_json_scalar(self, data: JSON, *, zarr_format: ZarrFormat) -> bytes:
 
         if check_json_str(data):
             return base64.standard_b64decode(data.encode("ascii"))
-        raise TypeError(f"Invalid type: {data}. Expected a string.")  # pragma: no cover
+        if check_json_byte_list(data):
+            return bytes(data)
+        raise TypeError(
+            f"Invalid type: {data}. Expected a base64-encoded string or a list of byte values."
+        )  # pragma: no cover
 
     def _check_scalar(self, data: object) -> TypeGuard[BytesLike]:
         """
diff --git a/src/zarr/core/dtype/npy/common.py b/src/zarr/core/dtype/npy/common.py
index f413f5f678..81941554e8 100644
--- a/src/zarr/core/dtype/npy/common.py
+++ b/src/zarr/core/dtype/npy/common.py
@@ -564,3 +564,22 @@ def check_json_bool(data: JSON) -> TypeGuard[bool]:
         True if the data is a boolean, False otherwise.
     """
     return isinstance(data, bool)
+
+
+def check_json_byte_list(data: JSON) -> TypeGuard[list[int]]:
+    """
+    Check if a JSON value is a list of byte values.
+
+    Parameters
+    ----------
+    data : JSON
+        The JSON value to check.
+
+    Returns
+    -------
+    Bool
+        True if the data is a list of integers in the range [0, 255], False otherwise.
+    """
+    return isinstance(data, list) and all(
+        isinstance(e, int) and not isinstance(e, bool) and 0 <= e <= 255 for e in data
+    )
diff --git a/tests/test_dtype/test_npy/test_bytes.py b/tests/test_dtype/test_npy/test_bytes.py
index 2d384d82ab..5662e6d253 100644
--- a/tests/test_dtype/test_npy/test_bytes.py
+++ b/tests/test_dtype/test_npy/test_bytes.py
@@ -2,6 +2,7 @@
 import pytest
 
 from tests.test_dtype.test_wrapper import BaseTestZDType
+from zarr.core.common import JSON
 from zarr.core.dtype.npy.bytes import NullTerminatedBytes, RawBytes, VariableLengthBytes
 from zarr.errors import UnstableSpecificationWarning
 
@@ -171,3 +172,38 @@ def test_invalid_size(zdtype_cls: type[NullTerminatedBytes] | type[RawBytes]) ->
     msg = f"length must be >= 1, got {length}."
     with pytest.raises(ValueError, match=msg):
         zdtype_cls(length=length)
+
+
+@pytest.mark.parametrize(
+    ("zdtype", "expected"),
+    [
+        (NullTerminatedBytes(length=2), np.bytes_(b"ab")),
+        (RawBytes(length=2), np.void(b"ab")),
+        (VariableLengthBytes(), b"ab"),
+    ],
+)
+def test_byte_list_fill_value(
+    zdtype: NullTerminatedBytes | RawBytes | VariableLengthBytes, expected: object
+) -> None:
+    """
+    Test that byte data types accept a JSON list of byte values as a fill value,
+    per the Zarr V3 spec, and that it matches the base64 form.
+    """
+    from_byte_list = zdtype.from_json_scalar([97, 98], zarr_format=3)
+    from_base64 = zdtype.from_json_scalar("YWI=", zarr_format=3)
+    assert from_byte_list == expected
+    assert from_byte_list == from_base64
+
+
+@pytest.mark.parametrize(
+    "zdtype", [NullTerminatedBytes(length=2), RawBytes(length=2), VariableLengthBytes()]
+)
+@pytest.mark.parametrize("data", [[256], [-1], ["a"], [97.0], [True]])
+def test_byte_list_fill_value_invalid(
+    zdtype: NullTerminatedBytes | RawBytes | VariableLengthBytes, data: JSON
+) -> None:
+    """
+    Test that a JSON list containing non-byte values is rejected as a fill value.
+    """
+    with pytest.raises(TypeError, match="byte values"):
+        zdtype.from_json_scalar(data, zarr_format=3)