MicroRNAs (miRNAs) are epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the role of miRNAs for replicative senescence of normal human keratinocytes (NHKs) remains unknown. Thus, we examined the expression profiles of 847 miRNAs in exponentially replicating and senescent NHKs and identified 126 senescence-associated miRNAs (SA-miRs). Among SA-miRs, 117 miRNAs (93%) were upregulated and 9 miRNAs (7%) were downregulated in senescent NHKs compared to those of exponentially replicating cells. Among the above miRNAs, we selected two miRNAs, miR-137 and miR-668, for further investigation because they were consistently upregulated with replicative senescence of three independent NHK cultures. Ectopic overexpression of miR-137 or miR-668 induced senescence in rapidly proliferating NHKs; a notable increase in senescence-associated β-galactosidase activity, p16INK4A and p53 was observed, indicating that they are novel senescence-inducing miRNAs. In addition, these senescence-inducing miRNAs were gradually increased during organismal aging of normal human oral epithelia. We also detected downregulation of miR-137 and miR-668 in many tested human head and neck squamous cell carcinoma cell lines. Since senescence would be viewed as a potent tumor suppressive pathway, the newly identified senescence-inducing miRNAs deserve to be further investigated for their therapeutic application in cancer treatment.