The brain is often described as an "immune-privileged" organ due to the presence of the blood-brain-barrier (BBB), which limits the entry of immune cells. In general, intracranial injection of adeno-associated virus (AAV) is considered a relatively safe procedure. In this study, we discovered that AAV, a popular engineered viral vector for gene therapy, can disrupt the BBB and induce immune cell infiltration in a titer-dependent manner. First, our bulk RNA sequencing data revealed that injection of high-titer AAV significantly upregulated many genes involved in disrupting BBB integrity and antiviral adaptive immune responses. By using histologic analysis, we further demonstrated that the biological structure of the BBB was severely disrupted in the adult mouse brain. Meanwhile, we noticed abnormal leakage of blood components, including immune cells, within the brain parenchyma of high-titer AAV injected areas. Moreover, we identified that the majority of infiltrated immune cells were cytotoxic T lymphocytes (CTLs), which resulted in a massive loss of neurons at the site of AAV injection. In addition, antagonizing CTL function by administering antibodies significantly reduced neuronal toxicity induced by high-titer AAV. Collectively, our findings underscore potential severe side effects of intracranial injection of high-titer AAV, which might compromise proper data interpretation if unaware of.