Vestibular hair cells are susceptible to damage from various stimuli such as infections, ischemia, and certain therapeutic drugs, including aminoglycoside antibiotics and the antineoplastic agent cisplatin. In mammals, damage to the vestibular hair cells is permanent. This study aimed to evaluate the protective effects of nobiletin (NOB) against aminoglycoside-induced hair cell death using utricles collected from adult mice. The utricles removed from CBA/N mice were assigned to eight groups according to the dose of NOB and the administration or not of neomycin. Hair cells in the utricles were counted by double labeling with calmodulin and calbindin. NOB inhibited hair cell death in utricles exposed to neomycin. The protective effect of NOB on hair cells in the utricles was also suggested to have resulted from the inhibition of the production and accumulation of 4-hydroxy-2-nonenal, the final product of lipid peroxide aldehyde. NOB suppressed neomycin-induced hair cell death. The principle of hair cell protection from aminoglycoside-induced hair cell death suggests that NOB inhibits reactive oxygen species formation in the utricles exposed to neomycin.