To investigate the effect of protein arginine methyltransferase 5 (PRMT5) selective inhibitor LLY-283 on cisplatin-induced inner ear hair cell injury, a cisplatin-induced HEI-OC1 cell injury model was first established. After the appropriate injury concentration was determined, the control group, cisplatin in-jury group and LLY-283 pretreatment group were set up. For the LLY-283 pretreatment group, the cells were pretreated with gradient concentrations of LLY-283 (40~140 μmol/L) for 2 h, and then treated with cisplatin for 24 h. The cell viability was detected by CCK-8 assay, and the apoptosis was observed by flow cytometry. Subsequently, an in vitro culture system of neonatal rat basement membrane was established and a zebrafish model was introduced to further verify the protective effect of LLY-283 on cisplatin-induced auditory hair cell damage. The results showed that, compared with the cisplatin injury group, the LLY-283 pretreatment group showed a significant reduction in cytotoxic effects of cisplatin, an increased survival rate of HEI-OC1 cells, and significant inhibition of the apoptosis induced by cisplatin. At the same time, LLY-283 reduced cisplatin-induced mortality of hair cells in the basement membrane of mice and also reduced cisplatin-in-duced damage to zebrafish lateral line hair cells. The preliminary experiments revealed that LLY-283 could antagonize the toxicity of cisplatin and protect auditory hair cells, indicating that the PRMT5 inhibitor may be a potential drug for the prevention and treatment of cisplatin-induced hearing loss.