Asiaticoside (AC) has been known to have anti-oxidative activity, however, the effect of AC on the progression of high glucose-induced hearing loss has not been studied. This study aims to analyze the effect of AC on cochlear hair cells under the treatment of high glucose in vitro and the hearing function in vivo . The results of MTT showed that high glucose decreased the activity of HEI-OC1 cells, but AC increased the activity of HEI-OC1 cells compared with high glucose group. The results of flow cytometry showed that AC decreased the degree of apoptosis induced by high levels of glucose. The results of DCFH-DA staining showed that AC inhibited the ROS production induced by high glucose levels. The results of JC-1 staining showed that AC inhibited the mitochondrial depolarization induced by high glucose levels. Furthermore, AC decreased the threshold, and protected inner and outer hair cells from damage in rats with hearing loss induced by diabetes mellitus. Moreover, AC decreased the activity of MDA, but, increased the activity of SOD, CAT and GSH-Px in vivo . AC also decreased the expression of AGEs, RAGE and NF-κB p65. Collectively, these results suggest that AC protects cochlear hair cells from high glucose-induced injury by increasing anti-oxidative activity and suppressing the AGEs/RAGE/NF-κB pathway.