diff --git a/newsfragments/5824.changed.md b/newsfragments/5824.changed.md
new file mode 100644
index 00000000000..3bcfc0afda2
--- /dev/null
+++ b/newsfragments/5824.changed.md
@@ -0,0 +1 @@
+Deprecate and remove `Py_TRACE_REFS` support (unsupported from Python 3.13).
\ No newline at end of file
diff --git a/pyo3-build-config/src/impl_.rs b/pyo3-build-config/src/impl_.rs
index 167013f5c42..a2e04c00df2 100644
--- a/pyo3-build-config/src/impl_.rs
+++ b/pyo3-build-config/src/impl_.rs
@@ -1147,6 +1147,7 @@ pub fn cross_compiling_from_cargo_env() -> Result<Option<CrossCompileConfig>> {
 pub enum BuildFlag {
     Py_DEBUG,
     Py_REF_DEBUG,
+    #[deprecated(since = "0.29.0", note = "no longer supported by PyO3")]
     Py_TRACE_REFS,
     Py_GIL_DISABLED,
     COUNT_ALLOCS,
@@ -1168,7 +1169,6 @@ impl FromStr for BuildFlag {
         match s {
             "Py_DEBUG" => Ok(BuildFlag::Py_DEBUG),
             "Py_REF_DEBUG" => Ok(BuildFlag::Py_REF_DEBUG),
-            "Py_TRACE_REFS" => Ok(BuildFlag::Py_TRACE_REFS),
             "Py_GIL_DISABLED" => Ok(BuildFlag::Py_GIL_DISABLED),
             "COUNT_ALLOCS" => Ok(BuildFlag::COUNT_ALLOCS),
             other => Ok(BuildFlag::Other(other.to_owned())),
@@ -1194,10 +1194,9 @@ impl FromStr for BuildFlag {
 pub struct BuildFlags(pub HashSet<BuildFlag>);
 
 impl BuildFlags {
-    const ALL: [BuildFlag; 5] = [
+    const ALL: [BuildFlag; 4] = [
         BuildFlag::Py_DEBUG,
         BuildFlag::Py_REF_DEBUG,
-        BuildFlag::Py_TRACE_REFS,
         BuildFlag::Py_GIL_DISABLED,
         BuildFlag::COUNT_ALLOCS,
     ];
diff --git a/pyo3-ffi/src/modsupport.rs b/pyo3-ffi/src/modsupport.rs
index b6186ddc8bc..e3dd1f2ee7c 100644
--- a/pyo3-ffi/src/modsupport.rs
+++ b/pyo3-ffi/src/modsupport.rs
@@ -83,7 +83,7 @@ pub const PYTHON_API_VERSION: i32 = 1013;
 pub const PYTHON_ABI_VERSION: i32 = 3;
 
 extern "C" {
-    #[cfg(not(py_sys_config = "Py_TRACE_REFS"))]
+
     #[cfg_attr(PyPy, link_name = "PyPyModule_Create2")]
     pub fn PyModule_Create2(module: *mut PyModuleDef, apiver: c_int) -> *mut PyObject;
 }
@@ -101,10 +101,7 @@ pub unsafe fn PyModule_Create(module: *mut PyModuleDef) -> *mut PyObject {
 }
 
 extern "C" {
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    fn PyModule_Create2TraceRefs(module: *mut PyModuleDef, apiver: c_int) -> *mut PyObject;
 
-    #[cfg(not(py_sys_config = "Py_TRACE_REFS"))]
     #[cfg_attr(PyPy, link_name = "PyPyModule_FromDefAndSpec2")]
     pub fn PyModule_FromDefAndSpec2(
         def: *mut PyModuleDef,
@@ -112,28 +109,6 @@ extern "C" {
         module_api_version: c_int,
     ) -> *mut PyObject;
 
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    fn PyModule_FromDefAndSpec2TraceRefs(
-        def: *mut PyModuleDef,
-        spec: *mut PyObject,
-        module_api_version: c_int,
-    ) -> *mut PyObject;
-}
-
-#[cfg(py_sys_config = "Py_TRACE_REFS")]
-#[inline]
-pub unsafe fn PyModule_Create2(module: *mut PyModuleDef, apiver: c_int) -> *mut PyObject {
-    PyModule_Create2TraceRefs(module, apiver)
-}
-
-#[cfg(py_sys_config = "Py_TRACE_REFS")]
-#[inline]
-pub unsafe fn PyModule_FromDefAndSpec2(
-    def: *mut PyModuleDef,
-    spec: *mut PyObject,
-    module_api_version: c_int,
-) -> *mut PyObject {
-    PyModule_FromDefAndSpec2TraceRefs(def, spec, module_api_version)
 }
 
 #[inline]
diff --git a/pyo3-ffi/src/object.rs b/pyo3-ffi/src/object.rs
index ddeabb9be3f..a6fc6d39ff6 100644
--- a/pyo3-ffi/src/object.rs
+++ b/pyo3-ffi/src/object.rs
@@ -96,10 +96,6 @@ const _PyObject_MIN_ALIGNMENT: usize = 4;
 #[cfg_attr(all(Py_3_15, Py_GIL_DISABLED), repr(C, align(4)))]
 #[derive(Debug)]
 pub struct PyObject {
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    pub _ob_next: *mut PyObject,
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    pub _ob_prev: *mut PyObject,
     #[cfg(Py_GIL_DISABLED)]
     pub ob_tid: libc::uintptr_t,
     #[cfg(all(Py_GIL_DISABLED, not(Py_3_14)))]
@@ -128,10 +124,6 @@ const _: () = assert!(std::mem::align_of::<PyObject>() >= _PyObject_MIN_ALIGNMEN
     reason = "contains atomic refcount on free-threaded builds"
 )]
 pub const PyObject_HEAD_INIT: PyObject = PyObject {
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    _ob_next: std::ptr::null_mut(),
-    #[cfg(py_sys_config = "Py_TRACE_REFS")]
-    _ob_prev: std::ptr::null_mut(),
     #[cfg(Py_GIL_DISABLED)]
     ob_tid: 0,
     #[cfg(all(Py_GIL_DISABLED, Py_3_15))]