Highlights•Peroxynitrite could induce Hair Cells (HCs) damage in vitro on mouse cochlear.•Peroxynitrite eventuated HCs loss in a basal-apical gradient manner.•Outer HCs were more vulnerable to peroxynitrite injury than inner HCs.•Peroxynitrite could cause mitochondria oxidative damage to HCs.•Low concentrations of Wnt3a have a protective effect on the oxidative damage of HCs.