Propolis is a natural resinous substance obtained from beehives, and emerging evidence supports that it has antitumor, antiinflammatory, antioxidant, and antimicrobial activities. The aim of the study is to examine the cytotoxic, antioxidant, and apoptotic features of ethanolic propolis extract (PE) on C6 glioma cells. The cells were treated with ethanolic PE at various concentrations for 24 hours, after which the total antioxidant status (TAS) and total oxidant status; malondialdehyde, protein carbonyl, 8-hydroxy-2′-deoxyguanosine, and glutathione (GSH) levels; Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity; and apoptotic markers were measured. Ethanolic PE at 100, 250, and 500 μg/mL concentrations showed optimal activity on C6 glioma cells. TAS and GSH levels were significantly increased in C6 glioma cells treated with 100 and 500 μg/mL PE compared to control cells (P < .05). Similarly, the activity of Cu/Zn-SOD was higher in C6 glioma cells treated with 250 or 500 μg/mL ethanolic PE compared to control cells (P < .05), as was the caspase-3 mRNA expression level. The highest levels of caspase-8 and -9 expression were in C6 glioma cells treated with 500 μg/mL PE. Collectively, our results indicate that ethanolic PE has cytotoxic and apoptotic effects on C6 glioma cells. Furthermore, it may provide a protective role in the antioxidant defense system. PE shows potential for development as a natural antioxidant and apoptotic agent for the treatment of brain tumors.© 2020 Wiley Periodicals, Inc.