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 that Lgr5+ 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 AAV-ie system to upregulate Rps14 expression in cultured hair cell progenitors and observed their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in vivo could promote supporting cells proliferation by activating the Wnt signaling pathway. In addition, over-expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing revealed showed that the neo- hair cells had transformed from Lgr5+ hair cell progenitors. Combined with RNA sequence analysis, signals regulated by Rps14 include cell cycle-related factors during supporting cell proliferation and Notch/TGF-β signaling during hair cell reprogramming. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.