Autophagy plays a key role in temozolomide (TMZ)-induced cytotoxicity in malignant glioma cells. TMZ, an 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 malignant glioma cells. Autophagy is marked by the recruitment of LC3 to autophagosome membranes. Inhibition of autophagy at an early stage with 3-methyladenine (3-MA) suppresses both LC3 localization and TMZ's antitumor effect. In contrast, bafilomycin A1, which inhibits autophagy at a later stage by preventing autophagosome-lysosome fusion, sensitizes tumor cells to TMZ by inducing apoptosis through caspase-3 activation, while LC3 localization remains unchanged. These findings suggest that TMZ induces autophagy in malignant glioma cells. Application of bafilomycin A1, which acts after LC3 associates with autophagosome membranes, enhances TMZ's cytotoxicity by inducing apoptosis. TMZ-induced autophagy may provide a self-defense mechanism for cancer cells, and inhibiting autophagy at a late stage, such as with bafilomycin A1, could enhance the antitumor effect of TMZ. The study highlights the importance of autophagy in cancer therapy and suggests that autophagy inhibitors like bafilomycin A1 may be promising candidates to combine with TMZ for improved treatment of malignant gliomas.Autophagy plays a key role in temozolomide (TMZ)-induced cytotoxicity in malignant glioma cells. TMZ, an 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 malignant glioma cells. Autophagy is marked by the recruitment of LC3 to autophagosome membranes. Inhibition of autophagy at an early stage with 3-methyladenine (3-MA) suppresses both LC3 localization and TMZ's antitumor effect. In contrast, bafilomycin A1, which inhibits autophagy at a later stage by preventing autophagosome-lysosome fusion, sensitizes tumor cells to TMZ by inducing apoptosis through caspase-3 activation, while LC3 localization remains unchanged. These findings suggest that TMZ induces autophagy in malignant glioma cells. Application of bafilomycin A1, which acts after LC3 associates with autophagosome membranes, enhances TMZ's cytotoxicity by inducing apoptosis. TMZ-induced autophagy may provide a self-defense mechanism for cancer cells, and inhibiting autophagy at a late stage, such as with bafilomycin A1, could enhance the antitumor effect of TMZ. The study highlights the importance of autophagy in cancer therapy and suggests that autophagy inhibitors like bafilomycin A1 may be promising candidates to combine with TMZ for improved treatment of malignant gliomas.