diff --git a/doc/source/whatsnew/v3.0.0.rst b/doc/source/whatsnew/v3.0.0.rst
index ec7b8b375abe5..e06df92a220dd 100644
--- a/doc/source/whatsnew/v3.0.0.rst
+++ b/doc/source/whatsnew/v3.0.0.rst
@@ -545,6 +545,7 @@ MultiIndex
 ^^^^^^^^^^
 - :func:`DataFrame.loc` with ``axis=0``  and :class:`MultiIndex` when setting a value adds extra columns (:issue:`58116`)
 - :meth:`DataFrame.melt` would not accept multiple names in ``var_name`` when the columns were a :class:`MultiIndex` (:issue:`58033`)
+- :meth:`MultiIndex.insert` would not insert NA value correctly at unified location of index -1 (:issue:`59003`)
 -
 
 I/O
diff --git a/pandas/core/indexes/multi.py b/pandas/core/indexes/multi.py
index 9d7c7f3e4a5c9..8e3ebc7816fed 100644
--- a/pandas/core/indexes/multi.py
+++ b/pandas/core/indexes/multi.py
@@ -3906,8 +3906,11 @@ def insert(self, loc: int, item) -> MultiIndex:
                 # have to insert into level
                 # must insert at end otherwise you have to recompute all the
                 # other codes
-                lev_loc = len(level)
-                level = level.insert(lev_loc, k)
+                if isna(k):  # GH 59003
+                    lev_loc = -1
+                else:
+                    lev_loc = len(level)
+                    level = level.insert(lev_loc, k)
             else:
                 lev_loc = level.get_loc(k)
 
diff --git a/pandas/tests/test_multilevel.py b/pandas/tests/test_multilevel.py
index 8f661edf0f241..e87498742061b 100644
--- a/pandas/tests/test_multilevel.py
+++ b/pandas/tests/test_multilevel.py
@@ -288,6 +288,13 @@ def test_multiindex_with_na(self):
 
         tm.assert_frame_equal(df, expected)
 
+    @pytest.mark.parametrize("na", [None, np.nan])
+    def test_multiindex_insert_level_with_na(self, na):
+        # GH 59003
+        df = DataFrame([0], columns=[["A"], ["B"]])
+        df[na, "B"] = 1
+        tm.assert_frame_equal(df[na], DataFrame([1], columns=["B"]))
+
 
 class TestSorted:
     """everything you wanted to test about sorting"""