We investigated whether the neuroprotective effect of estrogen is mediated by the estrogen receptor (ER) and whether extracellular signal-regulated kinase (ERK) is involved in the protective effect of estrogen against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. Retrograde labeling of retinal ganglion cells (RGCs) showed that pretreatment with 17β-estradiol (E2) using a silastic implant significantly attenuated the loss of RGCs induced by intravitreal injection of NMDA. Simultaneous administration of U0126, an ERK inhibitor, with NMDA completely abolished the protective effect of E2. Moreover, ICI182,780, an ER antagonist, also significantly diminished the protective effect of E2. Pretreatment with E2 significantly reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) 12 hr after NMDA injection. Moreover, ICI182,780 inhibited the ameliorative effect of E2 on TUNEL-positive cells in a dose-dependent manner. Immunostaining of anti-ERα monoclonal antibody was observed mainly in the RGCL and the INL. Western blot analysis showed a significant increase in the level of phosphorylated ERK (p-ERK) 6 hr after NMDA injection. However, NMDA did not increase the level of p-ERK protein 7 days after injection. Pretreatment of E2 induced further increases of p-ERK expression 6 hr and 7 days after NMDA injection, and U0126 and ICI182,780 significantly inhibited E2-induced p-ERK expression after 6 hr. These results suggest that E2 has an ER-mediated neuroprotective effect against NMDA-induced retinal neurotoxicity and that this effect may be associated with induction of p-ERK in the retina. © 2006 Wiley-Liss, Inc.