A major concern with regard to glioma treatment arises from the fact that high-grade gliomas are insensitive to the majority of anticancer therapies. The aim of the present study was to investigate antiproliferation potential of the BH3-mimetic gossypol in four glioma cells with different BRAF mutation status. Gossypol induced similar levels of growth inhibition in all glioma cell lines regardless of the BRAF mutation status. Glioma cells were found to be resistant to gossypol-mediated apoptosis; gossypol caused only a weak increase in the number of G2/M cells. However, gossypol treatment of glioma cells resulted in the progressive emergence of autophagic cells. In addition, the autophagy inhibitor 3-methyladenine (3-MA) partially rescued the growth inhibitory effect of gossypol on DBTRG-05MG cells, suggesting that pro-autophagic processes may be one of the underlying mechanisms for the sensitization of tumor cells to gossypol. Our data suggest that gossypol contributes to a more effective therapeutic strategy for brain tumor patients in which activation of apoptosis does not occur.