Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells. Anat Rec, 296:595–603, 2013. © 2013 Wiley Periodicals, Inc.