Neuroprotective effects of estrogens have been shown in various in vitro and in vivo models, but the mechanisms underlying protection by estrogen are not clear. Mounting evidence suggests antioxidant effects contribute to the neuroprotective effects of estrogens. In the present study, we assessed the protective effects of estrogens against H2O2-induced toxicity in human neuroblastoma cells and the potential mechanisms involved in this protection. We demonstrate that 17β-estradiol (17β-E2) increases cell survival against H2O2 toxicity in human neuroblastoma cells. 17β-E2 effectively reduced lipid peroxidation induced by 5-min H2O2 exposure. Furthermore, 17β-E2 exerts the protective effects by maintaining intracellular Ca2+ homeostasis, attenuating ATP depletion, ablating mitochondrial calcium overloading, and preserving mitochondrial membrane potential. Two nonfeminizing estrogens, 17α- and entestradiol, were as effective as 17β-E2 in increasing cell survival, alleviating lipid peroxidation, preserving mitochondrial function, and maintaining intracellular glutathione levels and Ca2+ homeostasis against H 2O2 insult. Moreover, the estrogen receptor antagonist fulvestrant (ICI 182,780) did not block effects of 17β-E2, but increased cell survival and blunted intracellular Ca2+ increases. However, these estrogens failed to reduce cytosolic reactive oxygen species, even at concentrations as high as 10 μM. In conclusion, estrogens exert protective effects against oxidative stress by inhibiting lipid peroxidation and subsequently preserving Ca2+ homeostasis, mitochondrial membrane potential, and ATP levels.