diff --git a/newsfragments/5169.misc.rst b/newsfragments/5169.misc.rst
new file mode 100644
index 0000000000..37f78c9b79
--- /dev/null
+++ b/newsfragments/5169.misc.rst
@@ -0,0 +1 @@
+Pre-process ``ext-module.define-macros`` from ``pyproject.toml`` into a list of tuples.
diff --git a/setuptools/config/_apply_pyprojecttoml.py b/setuptools/config/_apply_pyprojecttoml.py
index 2a59446042..225c693866 100644
--- a/setuptools/config/_apply_pyprojecttoml.py
+++ b/setuptools/config/_apply_pyprojecttoml.py
@@ -268,10 +268,18 @@ def _optional_dependencies(dist: Distribution, val: dict, _root_dir: StrPath | N
 def _ext_modules(dist: Distribution, val: list[dict]) -> list[Extension]:
     existing = dist.ext_modules or []
     args = ({k.replace("-", "_"): v for k, v in x.items()} for x in val)
-    new = [Extension(**kw) for kw in args]
+    new = (Extension(**_adjust_ext_attrs(kw)) for kw in args)
     return [*existing, *new]
 
 
+def _adjust_ext_attrs(attrs: dict) -> dict:
+    # https://github.com/pypa/setuptools/issues/4810
+    # In TOML there is no differentiation between tuples and lists,
+    # and distutils requires lists...
+    attrs["define_macros"] = list(map(tuple, attrs.get("define_macros") or []))
+    return attrs
+
+
 def _noop(_dist: Distribution, val: _T) -> _T:
     return val
 
diff --git a/setuptools/tests/config/test_apply_pyprojecttoml.py b/setuptools/tests/config/test_apply_pyprojecttoml.py
index 8f48c4316d..b6b21a3e9b 100644
--- a/setuptools/tests/config/test_apply_pyprojecttoml.py
+++ b/setuptools/tests/config/test_apply_pyprojecttoml.py
@@ -535,9 +535,14 @@ def test_invalid_module_name(self, tmp_path, monkeypatch, module):
 
 
 class TestExtModules:
+    def make_dist(self, toml_config):
+        pyproject = Path("pyproject.toml")
+        pyproject.write_text(cleandoc(toml_config), encoding="utf-8")
+        with pytest.warns(pyprojecttoml._ExperimentalConfiguration):
+            return pyprojecttoml.apply_configuration(Distribution({}), pyproject)
+
     def test_pyproject_sets_attribute(self, tmp_path, monkeypatch):
         monkeypatch.chdir(tmp_path)
-        pyproject = Path("pyproject.toml")
         toml_config = """
         [project]
         name = "test"
@@ -547,13 +552,28 @@ def test_pyproject_sets_attribute(self, tmp_path, monkeypatch):
           {name = "my.ext", sources = ["hello.c", "world.c"]}
         ]
         """
-        pyproject.write_text(cleandoc(toml_config), encoding="utf-8")
-        with pytest.warns(pyprojecttoml._ExperimentalConfiguration):
-            dist = pyprojecttoml.apply_configuration(Distribution({}), pyproject)
+        dist = self.make_dist(toml_config)
         assert len(dist.ext_modules) == 1
         assert dist.ext_modules[0].name == "my.ext"
         assert set(dist.ext_modules[0].sources) == {"hello.c", "world.c"}
 
+    def test_pyproject_define_macros_as_tuples(self, tmp_path, monkeypatch):
+        # https://github.com/pypa/setuptools/issues/4810
+        monkeypatch.chdir(tmp_path)
+        toml_config = """
+        [project]
+        name = "test"
+        version = "42.0"
+        [[tool.setuptools.ext-modules]]
+        name = "my.ext"
+        sources = ["hello.c", "world.c"]
+        define-macros = [["FIRST_SINGLE"], ["SECOND_TWO", "1"]]
+        """
+        dist = self.make_dist(toml_config)
+        assert isinstance(dist.ext_modules[0].define_macros[0], tuple)
+        assert dist.ext_modules[0].define_macros[0] == ("FIRST_SINGLE",)
+        assert dist.ext_modules[0].define_macros[1] == ("SECOND_TWO", "1")
+
 
 class TestDeprecatedFields:
     def test_namespace_packages(self, tmp_path):