S-nitrosylation is a redox-sensitive protein modification, which is a highly specific, but reversible mechanism that regulates several signal transduction cascades. Oxidative stress plays a causal role in the ototoxic effects of an anti-neoplastic drug, cisplatin. Despite emerging evidence implicating nitroxidative stress as a critical factor in cisplatin toxicity, the significance of the cochlear protein S-nitrosylation in cisplatin ototoxicity is yet to be understood. In the present study, a 16-mg/kg dose of cisplatin, induced a significant shift in the amplitudes of distortion product otoacoustic emissions, a measure of outer hair cell activity, in Wistar rats, 3 days post-treatment. These ototoxic effects were accompanied by significant increases in the S-nitrosylation of at least three cochlear proteins. Biological significance of these S-nitrosylated proteins was indicated by their immunolocalization in organ of Corti, stria vascularis, and spiral ganglions, which are known cochlear targets of cisplatin toxicity. In addition, co-treatment with Trolox, an inhibitor of peroxynitrite, attenuated cisplatin-induced S-nitrosylation of cochlear proteins and prevented the associated hearing loss. The cisplatin-induced S-nitrosylation of inner ear proteins, their sensitive cochlear localization, and their potential association with cisplatin-induced hearing loss suggests that S-nitrosylation of cochlear proteins might play a crucial role in mediating cisplatin ototoxicity.