Previous study reported that either selective GSK-3β inhibitor or up-regulating autophagy can alleviate cisplatin-induced ototoxicity. Other studies indicate that the activity of GSK-3β is closely associated with the autophagy level. The purpose of this study is to primarily explore the role of autophagy in the alleviation effect of GSK-3β inhibition on cisplatin-induced ototoxicity in vivo and in vitro. We observed the autophagy changes induced by GSK-3β inhibitor in outer hair cells (OHCs) in a cisplatin-induced ototoxicity rat model. In addition, autophagy inhibitor 3-MA was used in vitro experiments to observe the influence of autophagy inhibition on the cell protection effect due to GSK-3β inactivation. The relationship among autophagy, GSK-3β and cell damage were inferred. Negative regulation of GSK-3β significantly enhanced autophagy and alleviated cisplatin-induced hearing loss, OHC death in vivo and apoptosis in vitro. The autophagy inhibitor 3-MA inverted the protective effect of negative regulation of GSK-3β. These results indicated that enhancing autophagy may be a key downstream effect of GSK-3β inhibition in the alleviation of cisplatin-induced ototoxicity both in vivo and in vitro.