diff --git a/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher.ts b/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher.ts
index 1e0a87dc80f2..c8ba2baadefa 100644
--- a/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher.ts
+++ b/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher.ts
@@ -474,15 +474,34 @@ export function transformCalledOnceWith(
     return node;
   }
 
+  const expectCall = call.expression.expression;
+  const args = call.arguments;
+
+  // Splitting a negated assertion would assert that neither condition holds instead of not both.
+  if (ts.isPropertyAccessExpression(expectCall) && expectCall.name.text === 'not') {
+    reporter.reportTransformation(
+      sourceFile,
+      node,
+      'Transformed `.not.toHaveBeenCalledOnceWith()` to `.not.toHaveBeenCalledExactlyOnceWith()`.',
+    );
+
+    return ts.factory.updateExpressionStatement(
+      node,
+      ts.factory.updateCallExpression(
+        call,
+        createPropertyAccess(expectCall, 'toHaveBeenCalledExactlyOnceWith'),
+        call.typeArguments,
+        args,
+      ),
+    );
+  }
+
   reporter.reportTransformation(
     sourceFile,
     node,
     'Transformed `.toHaveBeenCalledOnceWith()` to `.toHaveBeenCalledTimes(1)` and `.toHaveBeenCalledWith()`.',
   );
 
-  const expectCall = call.expression.expression;
-  const args = call.arguments;
-
   const timesCall = ts.factory.createCallExpression(
     createPropertyAccess(expectCall, 'toHaveBeenCalledTimes'),
     undefined,
diff --git a/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher_spec.ts b/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher_spec.ts
index 274202ed1689..f123ac3b84b3 100644
--- a/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher_spec.ts
+++ b/packages/schematics/angular/refactor/jasmine-vitest/transformers/jasmine-matcher_spec.ts
@@ -113,6 +113,11 @@ describe('transformCalledOnceWith', () => {
           expect(mySpy).toHaveBeenCalledWith('foo', 'bar');
         `,
     },
+    {
+      description: 'should transform a negated toHaveBeenCalledOnceWith(...) into a single call',
+      input: `expect(mySpy).not.toHaveBeenCalledOnceWith('foo');`,
+      expected: `expect(mySpy).not.toHaveBeenCalledExactlyOnceWith('foo');`,
+    },
   ];
 
   testCases.forEach(({ description, input, expected }) => {