Gene therapy is proposed as a novel therapeutic strategy for treating glioblastoma multiforme (GBM), a devastating brain cancer. In the clinic, antivector immune responses pose formidable challenges. Herein we demonstrate that high-capacity adenovirus vectors (HC-Ads) carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase (TK) induce tumor regression and long-term survival in an intracranial glioma model, even in the presence of systemic antiadenovirus immunity, as could be encountered in patients. First-generation Ad-TK failed to elicit tumor regression in this model. These results pave the way for implementing HC-Ad-TK-mediated gene therapy as a powerful adjuvant for treating GBM.