Gentamicin is a well-known ototoxic aminoglycoside. However, the mechanism underlying this ototoxicity remains unclear. One of the mechanisms which may be responsible for this ototoxicity is excitotoxic damage to hair cells. The overstimulation of the N-methyl-d-aspartate (NMDA) receptors increases the production of nitric oxide (NO), which induces oxidative stress on hair cells. In order to determine the mechanism underlying this excitotoxicity, we treated guinea pigs with gentamicin by placing gentamicin (0.5 mg) pellets into a round window niche. After the sacrifice of the animals, which occurred at 3, 7 and 14 days after the treatment, the numbers of hair cells in the animals were counted with a scanning electron microscope. We then performed immunostaining using neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS) and nitrotyrosine antibodies. The number of hair cells in the animals was found to decrease significantly after 7 days. nNOS and iNOS expression levels were observed to have increased 3 days after treatment. Nitrotyrosine was expressed primarily at the calyceal afferents of the type I hair cells 3 days after treatment. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining revealed positive hair cells 3 days after treatment. Our results suggest that inner ear treatment with gentamicin may upregulate nNOS and iNOS to induce oxidative stress in the calyceal afferents of type I hair cells, via nitric oxide overproduction.