Although many studies have suggested that estrogen acts as a neuroprotective agent in oxidative stress, the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of 17-estradiol (17-E2) on H₂O₂-induced death signaling in cultured cortical neurons. Exposure of the cortical neurons to H₂O₂ triggered a series of events including over-activation of p44/42 mitogen-activated protein kinase (MAPK) and intracellular Ca²⁺ accumulation via voltage-gated Ca²⁺ channels and ionotropic glutamate receptors, resulting in apoptotic-like cell death. The MAPK pathway might work as death signaling in our system, as the MAPK pathway inhibitor, U0126, blocked H₂O₂-induced MAPK activation, Ca²⁺ overload, and cell death. Interestingly, a similar inhibitory effect on H₂O₂-triggered MAPK activation, Ca²⁺ accumulation, and cell death was observed in cultures incubated with 17-E2 for 24 h before exposure to H₂O₂, suggesting that the protective effect of 17-E2 is induced via attenuating over-activation of the MAPK pathway. Furthermore, we found that ionotropic glutamate receptor subunits including NR2A and GluR2/3, but not NR2B and GluR1, were down-regulated in the 17-E2-treated cultures. The down-regulation of these glutamate receptor subunits was also observed after chronic treatment with U0126. Therefore, it is possible that 17-E2 down-regulates the expression of the ionotropic glutamate receptors by reducing activity of the MAPK pathway, which might be important for the protective effect of 17-E2 against oxidative stress-induced toxicity.