Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.