Elevation of p16(INK4a) has been described as an important mechanism for hydrogen peroxide (H2O2)-induced replicative senescence. However, the mechanisms underlying remain unknown. In this study, we demonstrate an important role of RNA-binding protein AUF1-mediated mRNA turnover in H2O2-induced p16(INK4a) expression. The induction of p16 by H2O2 was accompanied with declined cytoplasmic AUF1 level. Accordingly, exposure of cells to H2O2 remarkably reduced the binding of AUF1 to p16 3'UTR and increased the half-life of an EGFP-p16-3'UTR chimeric transcript. In AUF1-silenced cells, the effect of H2O2 on p16 induction was abolished. Furthermore, in cells co-transfected with vectors expressing AUF1s, treatment with H2O2 failed to significantly reduce the expression of AUF1 and subsequently elevate the levels of p16. Moreover, HeLa cells overexpressing AUF1s were resistant to H2O2-induced senescence. Our results indicate that AUF1 is critical for H2O2-induced p16 expression and cellular senescence.