AbstractSensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown thatLgr5+supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno‐associated virus‐inner ear system to upregulateRps14expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly,Rps14overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over‐expressingRps14induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+progenitors. In conclusion, our analysis reveals the potential role ofRps14in driving hair cell regeneration in mammalian.