To investigate the role of glutamate transporters (GLTs)in retinal glial cells that were treated with N-methyl-D-aspartate (NMDA), in retinal ganglion cell (RGC) survival. Primary cultures of retinal glial cells or RGCs from 3-day-old Sprague-Dawley rats were employed in the present study. Retinal glial cells were treated with NMDA and changes in GLT mRNA and protein expression were analyzed. The effects of pretreating retinal glial cells with the GLAST-specific inhibitor, rottlerin (ROT), and the GLT-1-specific inhibitor, dihydrokainic acid (DHK), on RGC survival were investigated under exposure to NMDA. The amount of glutamate in the culture medium of retinal glial cells was measured by high-performance liquid chromatography. NMDA treatment increased GLAST and GLT-1 expression. GLAST and GLT-1 mRNA expression increased by 2.94-fold and 3.36-fold at 12 h after treatment with the highest concentration of NMDA (33 mM), and by 1.41-fold and 1.39-fold at 24 h, respectively. GLT-1 and GLAST protein expression also increased. MK801, an NMDA-receptor antagonist, inhibited the NMDA-induced upregulation of GLT mRNA expression. Co-culture with retinal glial cells increased the survival rate of RGCs. ROT decreased the survival rate of RGCs, whereas DHK significantly increased the survival rate of RGCs treated with 33 mM NMDA. NMDA treatment reduced the total amount of glutamate in the culture medium, particularly when 33 mM NMDA was added to the medium. ROT pretreatment increased the amount of glutamate in the culture medium, whereas DHK pretreatment decreased it. GLAST and GLT-1 may have different roles in the survival of RGCs mediated by retinal glial cells. These results suggest that the NMDA-associated induction of GLTs plays an important role in RGC survival.