Oncolytic adenoviruses are a promising tool in cancer therapy. In this study, we characterized the role of autophagy in oncolytic adenovirus-induced therapeutic effects. OBP-405, an oncolytic adenovirus regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad, OBP-301) with a tropism modification (RGD) exhibited a strong antitumor effect on glioblastoma cells. When autophagy was inhibited pharmacologically, the cytotoxicity of OBP-405 was attenuated. In addition, autophagy-deficient Atg5(-/-) mouse embryonic fibroblasts (MEFs) were less sensitive than wild-type MEFs to OBP-405. These findings indicate that OBP-405-induced autophagy is a cell killing effect. Moreover, autophagy-inducing therapies (temozolomide and rapamycin) synergistically sensitized tumor cells to OBP-405 by stimulating the autophagic pathway without altering OBP-405 replication. Mice harboring intracranial tumors treated with OBP-405 and temozolomide survived significantly longer than those treated with temozolomide alone, and mice treated with OBP-405 and the rapamycin analog RAD001 survived significantly longer than those treated with RAD001 alone. The observation that autophagy inducers increase OBP-405 antitumor activity suggests a novel strategy for treating patients with glioblastoma.