diff --git a/crates/ruff_linter/resources/test/fixtures/flake8_pyi/PYI016.py b/crates/ruff_linter/resources/test/fixtures/flake8_pyi/PYI016.py
index 89efd61e8f..74eaf356d5 100644
--- a/crates/ruff_linter/resources/test/fixtures/flake8_pyi/PYI016.py
+++ b/crates/ruff_linter/resources/test/fixtures/flake8_pyi/PYI016.py
@@ -142,3 +142,7 @@ field47: typing.Optional[int] | typing.Optional[dict]
 # avoid reporting twice
 field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 field49: typing.Optional[complex | complex] | complex
+
+# Regression test for https://github.com/astral-sh/ruff/issues/19403
+# Should throw duplicate union member but not fix
+isinstance(None, typing.Union[None, None])
\ No newline at end of file
diff --git a/crates/ruff_linter/src/rules/flake8_pyi/rules/duplicate_union_member.rs b/crates/ruff_linter/src/rules/flake8_pyi/rules/duplicate_union_member.rs
index 33812cebee..2fefb8e01d 100644
--- a/crates/ruff_linter/src/rules/flake8_pyi/rules/duplicate_union_member.rs
+++ b/crates/ruff_linter/src/rules/flake8_pyi/rules/duplicate_union_member.rs
@@ -64,6 +64,7 @@ pub(crate) fn duplicate_union_member<'a>(checker: &Checker, expr: &'a Expr) {
     let mut diagnostics = Vec::new();
 
     let mut union_type = UnionKind::TypingUnion;
+    let mut optional_present = false;
     // Adds a member to `literal_exprs` if it is a `Literal` annotation
     let mut check_for_duplicate_members = |expr: &'a Expr, parent: &'a Expr| {
         if matches!(parent, Expr::BinOp(_)) {
@@ -74,6 +75,7 @@ pub(crate) fn duplicate_union_member<'a>(checker: &Checker, expr: &'a Expr) {
             && is_optional_type(checker, expr)
         {
             // If the union member is an `Optional`, add a virtual `None` literal.
+            optional_present = true;
             &VIRTUAL_NONE_LITERAL
         } else {
             expr
@@ -87,7 +89,7 @@ pub(crate) fn duplicate_union_member<'a>(checker: &Checker, expr: &'a Expr) {
                 DuplicateUnionMember {
                     duplicate_name: checker.generator().expr(virtual_expr),
                 },
-                // Use the real expression's range for diagnostics,
+                // Use the real expression's range for diagnostics.
                 expr.range(),
             ));
         }
@@ -104,6 +106,13 @@ pub(crate) fn duplicate_union_member<'a>(checker: &Checker, expr: &'a Expr) {
         return;
     }
 
+    // Do not reduce `Union[None, ... None]` to avoid introducing a `TypeError` unintentionally
+    // e.g. `isinstance(None, Union[None, None])`, if reduced to `isinstance(None, None)`, causes
+    // `TypeError: isinstance() arg 2 must be a type, a tuple of types, or a union` to throw.
+    if unique_nodes.iter().all(|expr| expr.is_none_literal_expr()) && !optional_present {
+        return;
+    }
+
     // Mark [`Fix`] as unsafe when comments are in range.
     let applicability = if checker.comment_ranges().intersects(expr.range()) {
         Applicability::Unsafe
diff --git a/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__PYI016_PYI016.py.snap b/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__PYI016_PYI016.py.snap
index 8a51694b4a..62c0d69f78 100644
--- a/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__PYI016_PYI016.py.snap
+++ b/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__PYI016_PYI016.py.snap
@@ -974,6 +974,8 @@ PYI016.py:143:61: PYI016 [*] Duplicate union member `complex`
 143     |-field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
     143 |+field48: typing.Union[typing.Optional[complex], complex]
 144 144 | field49: typing.Optional[complex | complex] | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
 
 PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
     |
@@ -981,6 +983,8 @@ PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144 | field49: typing.Optional[complex | complex] | complex
     |                                    ^^^^^^^ PYI016
+145 |
+146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
     |
     = help: Remove duplicate union member `complex`
 
@@ -990,3 +994,15 @@ PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
 143 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144     |-field49: typing.Optional[complex | complex] | complex
     144 |+field49: typing.Optional[complex] | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
+147 147 | # Should throw duplicate union member but not fix
+
+PYI016.py:148:37: PYI016 Duplicate union member `None`
+    |
+146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
+147 | # Should throw duplicate union member but not fix
+148 | isinstance(None, typing.Union[None, None])
+    |                                     ^^^^ PYI016
+    |
+    = help: Remove duplicate union member `None`
diff --git a/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__preview__PYI016_PYI016.py.snap b/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__preview__PYI016_PYI016.py.snap
index 8b49997fcc..a1212a7b5b 100644
--- a/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__preview__PYI016_PYI016.py.snap
+++ b/crates/ruff_linter/src/rules/flake8_pyi/snapshots/ruff_linter__rules__flake8_pyi__tests__preview__PYI016_PYI016.py.snap
@@ -1162,6 +1162,8 @@ PYI016.py:143:61: PYI016 [*] Duplicate union member `complex`
 143     |-field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
     143 |+field48: typing.Union[None, complex]
 144 144 | field49: typing.Optional[complex | complex] | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
 
 PYI016.py:143:72: PYI016 [*] Duplicate union member `complex`
     |
@@ -1179,6 +1181,8 @@ PYI016.py:143:72: PYI016 [*] Duplicate union member `complex`
 143     |-field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
     143 |+field48: typing.Union[None, complex]
 144 144 | field49: typing.Optional[complex | complex] | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
 
 PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
     |
@@ -1186,6 +1190,8 @@ PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144 | field49: typing.Optional[complex | complex] | complex
     |                                    ^^^^^^^ PYI016
+145 |
+146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
     |
     = help: Remove duplicate union member `complex`
 
@@ -1195,6 +1201,9 @@ PYI016.py:144:36: PYI016 [*] Duplicate union member `complex`
 143 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144     |-field49: typing.Optional[complex | complex] | complex
     144 |+field49: None | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
+147 147 | # Should throw duplicate union member but not fix
 
 PYI016.py:144:47: PYI016 [*] Duplicate union member `complex`
     |
@@ -1202,6 +1211,8 @@ PYI016.py:144:47: PYI016 [*] Duplicate union member `complex`
 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144 | field49: typing.Optional[complex | complex] | complex
     |                                               ^^^^^^^ PYI016
+145 |
+146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
     |
     = help: Remove duplicate union member `complex`
 
@@ -1211,3 +1222,15 @@ PYI016.py:144:47: PYI016 [*] Duplicate union member `complex`
 143 143 | field48: typing.Union[typing.Optional[typing.Union[complex, complex]], complex]
 144     |-field49: typing.Optional[complex | complex] | complex
     144 |+field49: None | complex
+145 145 | 
+146 146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
+147 147 | # Should throw duplicate union member but not fix
+
+PYI016.py:148:37: PYI016 Duplicate union member `None`
+    |
+146 | # Regression test for https://github.com/astral-sh/ruff/issues/19403
+147 | # Should throw duplicate union member but not fix
+148 | isinstance(None, typing.Union[None, None])
+    |                                     ^^^^ PYI016
+    |
+    = help: Remove duplicate union member `None`