MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.