In this study, we investigated the effects of G-protein coupled estrogen receptor (GPER) activation in the early phase of retinopathy of prematurity (ROP) and its association with endoplasmic reticulum (ER) stress using primary murine retinal microglia as an experimental model. Fluorescence microscopy results show that the CD11c-positive primary retinal microglia in vitro cultured for 14 days were GPER-positive. GPER activation using GPER-agonist G-1 reduced autophagy and increased the viability of the hyperoxia-treated primary murine retinal microglia. Furthermore, GPER activation reduced the expression of ER stress-related proteins, IRE1α, PERK and ATF6 in the hyperoxia-treated primary murine retinal microglia compared to the corresponding controls. GPER activation significantly reduced a time-dependent increase in IP3R-dependent calcium release from the ER, thereby maintaining higher calcium levels in the ER of hyperoxia-treated primary retinal microglia. However, the protective effects of G-1 on the hyperoxia-treated primary retinal microglia were eliminated by inactivation of GPER using the GPER-antagonist, G-15. In conclusion, our study demonstrates that GPER activation enhances the survival of hyperoxia-treated primary retinal microglia by reducing ER stress. Our study demonstrates the therapeutic potential of GPER agonists such as G-1 in the early phase of ROP.