Breast cancer brain metastases (BCBM) are a deadly sequela of breast tumors that overexpress human epidermal growth factor receptor 2 (HER2). HER2+ BCBM affects approximately 17,000 women in the US every year. Median survival is 10-13 months from the time of diagnosis of central nervous system (CNS) disease. Current therapeutic interventions are invasive, toxic, and largely ineffective, leaving a clear, unmet need for targeted HER2+ BCBM treatments. Trastuzumab(Herceptin®) is a monoclonal antibody used to treat HER2+ breast cancer successfully, but systemic trastuzumab cannot bypass the blood-brain barrier (BBB). To solve this problem, we have developed an adeno-associated virus serotype 9 (AAV9) vector to express trastuzumab in vivo after a single intrathecal (IT) injection. IT vector administration in an orthotopic Rag1-/- murine xenograft model of HER2+ BCBM led to a significant increase in median survival and attenuated brain tumor growth. We also report preservation of both the HER2 antigen specificity and the natural killer (NK) cell-associated mechanism of action of trastuzumab. Finally, we demonstrate increased median survival when IT AAV9.trastuzumab is administered as tumor treatment. Our results indicate that IT AAV9.trastuzumab may provide significant anti-tumor activity in patients with HER2+ BCBM.