Cisplatin-induced ototoxicity is caused by reactive oxygen species. It has been recognized that estradiol (E2) regulates redox balance. However, little is known about the protective mechanisms of E2 against cisplatin-induced ototoxicity. In this study, we investigated the effect of E2 on nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated hair cell protection using the organ of Corti isolated from mice. The organ of Corti collected from C57BL/6 mice at 3–5 postnatal days was used in all experiments. The organ of Corti was exposed to 20 μM cisplatin with/without 100 nM E2 to examine the effect of E2 on cisplatin-induced hair cell loss. The mRNA expression of Nrf2 and the phase II detoxification gene after E2 and cisplatin treatment was analyzed using quantitative real-time PCR. E2 significantly reduces cisplatin-induced cochlear hair cell death. In addition, 100 nM E2 increased the mRNA expression of Nrf2 and phase II detoxification genes in the organ of Corti under cisplatin treatment. Our results suggest that E2 activates Nrf2, phase II detoxification enzymes and exerts a protective effect against cisplatin-induced ototoxicity.