This study investigates the role of autophagy in the cytotoxicity of temozolomide (TMZ) against malignant glioma cells. TMZ, a new alkylating agent, inhibits the viability of malignant glioma cells in a dose-dependent manner and induces G2/M arrest. At a clinically achievable dose (100 μM), TMZ induces autophagy but not apoptosis in these cells. The microtubule-associated protein light-chain 3 (LC3), a key autophagy marker, is recruited to autophagosome membranes after TMZ treatment. Inhibiting autophagy with 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, suppresses the antitumor effect of TMZ by preventing LC3 localization to autophagosomes. Conversely, bafilomycin A1, a specific inhibitor of vacuolar-type H^+^-ATPase, sensitizes tumor cells to TMZ by inducing apoptosis through activation of caspase-3 and mitochondrial and lysosomal membrane permeabilization. These findings suggest that TMZ-induced autophagy is a critical component of its cytotoxicity, and inhibiting this process with bafilomycin A1 enhances the antitumor effect of TMZ.This study investigates the role of autophagy in the cytotoxicity of temozolomide (TMZ) against malignant glioma cells. TMZ, a new alkylating agent, inhibits the viability of malignant glioma cells in a dose-dependent manner and induces G2/M arrest. At a clinically achievable dose (100 μM), TMZ induces autophagy but not apoptosis in these cells. The microtubule-associated protein light-chain 3 (LC3), a key autophagy marker, is recruited to autophagosome membranes after TMZ treatment. Inhibiting autophagy with 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, suppresses the antitumor effect of TMZ by preventing LC3 localization to autophagosomes. Conversely, bafilomycin A1, a specific inhibitor of vacuolar-type H^+^-ATPase, sensitizes tumor cells to TMZ by inducing apoptosis through activation of caspase-3 and mitochondrial and lysosomal membrane permeabilization. These findings suggest that TMZ-induced autophagy is a critical component of its cytotoxicity, and inhibiting this process with bafilomycin A1 enhances the antitumor effect of TMZ.