diff --git a/changelog/3225.bugfix.rst b/changelog/3225.bugfix.rst
new file mode 100644
index 00000000000..9985da918e7
--- /dev/null
+++ b/changelog/3225.bugfix.rst
@@ -0,0 +1 @@
+A non-autouse fixture overriding an autouse one no longer runs automatically, without changing explicit fixture requests.
diff --git a/src/_pytest/fixtures.py b/src/_pytest/fixtures.py
index 7656fca2f5b..118557e3c76 100644
--- a/src/_pytest/fixtures.py
+++ b/src/_pytest/fixtures.py
@@ -1124,7 +1124,7 @@ def __init__(
         ids: tuple[object | None, ...] | Callable[[Any], object | None] | None = None,
         *,
         node: nodes.Node | NotSetType = NOTSET,
-        # only used in a deprecationwarning msg, can be removed in pytest9
+        # Whether the fixture is autouse; consulted during fixture closure (#3225).
         _autouse: bool = False,
         _ispytest: bool = False,
     ) -> None:
@@ -1186,7 +1186,7 @@ def __init__(
         self.cached_result: _FixtureCachedResult[FixtureValue] | None = None
         self._finalizers: Final[list[Callable[[], object]]] = []
 
-        # only used to emit a deprecationwarning, can be removed in pytest9
+        # Whether the fixture is autouse; consulted during fixture closure (#3225).
         self._autouse = _autouse
 
     @property
@@ -1938,14 +1938,27 @@ def pytest_collection_finish(self) -> None:
 
     def _getautousenames(self, node: nodes.Node) -> Iterator[str]:
         """Return the names of autouse fixtures visible to node."""
+        usefixtures_ini = set(self.config.getini("usefixtures"))
         for parentnode in node.listchain():
             basenames = self._node_autousenames.get(parentnode)
             if basenames:
-                yield from basenames
-            # Legacy fallback: check string-based nodeid autouse names.
+                for name in basenames:
+                    if name in usefixtures_ini or self._is_autouse(name, node):
+                        yield name
+            # Legacy fallback: string-based nodeid autouse names.
             nodeid_basenames = self._nodeid_autousenames.get(parentnode.nodeid)
             if nodeid_basenames:
-                yield from nodeid_basenames
+                for name in nodeid_basenames:
+                    if self._is_autouse(name, node):
+                        yield name
+
+    def _is_autouse(self, name: str, node: nodes.Node) -> bool:
+        """Whether the fixture resolved for name is itself autouse.
+
+        A non-autouse override cancels the autouse fixture it shadows (#3225).
+        """
+        fixturedefs = self.getfixturedefs(name, node)
+        return not fixturedefs or fixturedefs[-1]._autouse
 
     def _getusefixturesnames(self, node: nodes.Item) -> Iterator[str]:
         """Return the names of usefixtures fixtures visible to node."""
diff --git a/testing/python/fixtures.py b/testing/python/fixtures.py
index 8e779a93d76..9d599216db9 100644
--- a/testing/python/fixtures.py
+++ b/testing/python/fixtures.py
@@ -2560,6 +2560,112 @@ def test_hello(arg1):
         reprec = pytester.inline_run()
         reprec.assertoutcome(passed=1)
 
+    def test_autouse_cancelled_by_non_autouse_override(
+        self, pytester: Pytester
+    ) -> None:
+        """A non-autouse override cancels the autouse fixture it shadows (#3225)."""
+        pytester.makeconftest(
+            """
+            import pytest
+
+            @pytest.fixture(autouse=True)
+            def foo():
+                pass
+            """
+        )
+        pytester.makepyfile(
+            """
+            import pytest
+
+            @pytest.fixture()
+            def foo():
+                assert False
+
+            def test_bar(foo):
+                pass
+
+            def test_baz():
+                pass
+            """
+        )
+        result = pytester.runpytest()
+        result.assert_outcomes(passed=1, errors=1)
+        result = pytester.runpytest("-o", "usefixtures=foo")
+        result.assert_outcomes(errors=2)
+
+    def test_autouse_cancelled_by_non_autouse_class_override(
+        self, pytester: Pytester
+    ) -> None:
+        """A non-autouse class-level override cancels the autouse fixture (#3225)."""
+        pytester.makepyfile(
+            """
+            import pytest
+
+            @pytest.fixture(autouse=True)
+            def foo():
+                pass
+
+            class TestClass:
+                @pytest.fixture()
+                def foo(self):
+                    assert False
+
+                def test_with_request(self, foo):
+                    pass
+
+                def test_no_request(self):
+                    pass
+
+            def test_module_level():
+                pass
+            """
+        )
+        result = pytester.runpytest()
+        result.assert_outcomes(passed=2, errors=1)
+
+    def test_getautousenames_legacy_nodeid_autouse(self, pytester: Pytester) -> None:
+        """Autouse registered via the deprecated nodeid API is still yielded."""
+        pytester.makeconftest(
+            """
+            import pytest
+
+            @pytest.fixture
+            def fm(request):
+                return request._fixturemanager
+
+            @pytest.fixture
+            def item(request):
+                return request._pyfuncitem
+            """
+        )
+        pytester.makepyfile(
+            """
+            import warnings
+
+            def test_legacy(item, fm):
+                with warnings.catch_warnings():
+                    warnings.simplefilter("ignore")
+                    fm._register_fixture(
+                        name="legacy_auto",
+                        func=lambda: None,
+                        nodeid=item.nodeid,
+                        autouse=True,
+                    )
+                assert "legacy_auto" in list(fm._getautousenames(item))
+                with warnings.catch_warnings():
+                    warnings.simplefilter("ignore")
+                    fm._register_fixture(
+                        name="legacy_auto",
+                        func=lambda: None,
+                        nodeid=item.nodeid,
+                        autouse=False,
+                    )
+                assert "legacy_auto" not in list(fm._getautousenames(item))
+            """
+        )
+        reprec = pytester.inline_run()
+        reprec.assertoutcome(passed=1)
+
     @pytest.mark.parametrize("param1", ["", "params=[1]"], ids=["p00", "p01"])
     @pytest.mark.parametrize("param2", ["", "params=[1]"], ids=["p10", "p11"])
     def test_ordering_dependencies_torndown_first(