Abnormal development of immature retinal vascular structure in preterm infants under the condition of hyperoxia is the primary cause of retinopathy of prematurity (ROP), which has become the leading cause of blindness in children. Retinal ganglion cells (RGCs) play a critical role in the normal growth of retinal vessels. Previous studies have indicated that estrogen can alleviate retinal lesions in the ROP animal model by inhibiting reactive oxygen species, which is associated with endoplasmic reticulum (ER) stress. This study aimed to investigate the protecting effect of G-protein coupled estrogen receptor (GPER), one of the estrogen receptors distributed in ER, on RGCs in the early stage of ROP and its relationship with ER stress. We found that GPER was widely expressed in primary cultured murine RGCs. GPER activation by its agonist G-1 increased cell vitality and decreased apoptosis and autophagy of RGCs under hyperoxia. GPER activation by G-1 decreased the expressions of the ER stress proteins, including inositol-requiring kinase/endonuclease 1α, pancreatic ER stress kinase, and cleaved activating transcription factor 6 in ER of RGCs under hyperoxia. GPER activation decreased IP3R activity and increased Ca concentration in ER of RGCs under hyperoxia. In addition, GPER antagonist (G-15) reversed all these effects of the GPER agonist mentioned above. This study suggested that GPER activation can protect the survival of RGCs in the early stage of ROP via reducing ER stress in RGCs under the condition of hyperoxia.© 2020 Wiley Periodicals LLC.