OBJECTIVE To explore the protective effect of 2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucosid(TSG) on hearing injury in acetaminophen(APAP) induced guinea pigs. METHODS The ear injury of guinea pigs was induced by APAP, after treatment with TSG, the auditory brainstem response(ABR) and distortion product otoacoustic emission(DPOAE) of guinea pigs were tested to judge the hearing loss, the injury of cochlear hair cells was observed by immunofluorescence, and the liver and kidney tissues of guinea pigs were observed by pathology to judge the toxicity of TSG to liver and kidney. The damage of HEI-OC1 auditory nerve cells induced by APAP was used as the study of ear injury cells. The cell survival and apoptosis were detected by MTT method, cell morphology observation, and flow cytometry, the expression of Birc5 gene was detected by fluorescence quantitative PCR. RESULTS Compared with the model group, on the 14th day, the ABR hearing threshold of guinea pigs decreased significantly, the difference was more significant when the DPOAE test frequency was 2 kHz(P<0.01). The acoustic startle reflex test showed that the amplitude of startle reflex decreased significantly at 100 dB(P<0.05). The gap-prepulse inhibition experiment showed that the amplitude of startle reflex decreased at 10, 40, 50 ms. The guinea pig cochlea was peeled off and the integrity of hair cells was observed after immunofluorescence staining. Compared with the model group, the outer hair cells in TSG group were arranged orderly without loss, and the inner hair cells were basically arranged orderly. Pathological observation showed that TSG had certain hepatotoxicity, but the nephrotoxicity was not obvious. The cell experiment found that the half inhibitory concentration of APAP on HEI-OC1 auditory nerve cells was 800 µmol·L-1. When 10 µmol·L-1 TSG intervened in APAP induced cell injury, the apoptosis rate decreased to 16.0%, which was significantly lower than 25.5% in model(APAP) group. In APAP group, the expression level of Birc5 in HEI-OC1 cells decreased significantly, and TSG treatment could significantly increase the expression level of Birc5. CONCLUSION TSG 15 mg·kg-1 can protect guinea pigs from hearing loss induce by 100 mg·kg-1APAP for 14 d. The protective effect is related to TSG inhibiting APAP induces auditory nerve cell apoptosis and promoting the expression of apoptosis inhibitor Birc5.