N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from flesh fly, has been described as having multipotential biological activities toward various tissues. However, there has been no report testing its action on neural cells. In the present study, we investigate whether 5-S-GAD is neurotoxic or neuroprotective to the rat retina. 5-S-GAD at high doses (more than 200 pmol) induced apoptosis of retinal neurons 7 days after intraocular injection. 5-S-GAD at low doses (2-20 pmol) significantly attenuated the loss of retinal ganglion cells (RGCs) and the thinning of inner retina induced by NMDA in a dose-dependent manner. To understand the protective mechanism of 5-S-GAD, we investigated the influence of 5-S-GAD on the cell survival molecules, phospho-Akt and Bcl-2. 5-S-GAD (2-20 pmol) rapidly increased phospho-Akt expression 1-7 days and Bcl-2 expression 3-7 days after injection. The cellular localization of this increase was both in bipolar cells and RGCs. This neurosurvival effect of 5-S-GAD was further tested using another model of optic nerve injury. 5-S-GAD significantly blocked the apoptosis of RGCs 7 days after optic nerve crush. These results show that 5-S-GAD (2-20 pmol) protects against the NMDA- and optic nerve crush-induced apoptosis of RGCs. The neuroprotective action of 5-S-GAD in the retina might be mediated by the cell survival phospho-Akt/Bcl-2 system and offers a therapeutic option to rescue RGCs from various types of excitotoxic disease, such as glaucoma.