Hyperglycemia-mediated damage to retinal pigment epithelial (RPE) cells plays a central role in the pathogenesis of diabetic retinopathy. Dysregulation of microRNA (miR)-383 modulates pancreatic beta cell survival in diabetes; however, its role in diabetic retinopathy remains unclear. In this study, we examined the expression of miR-383 in ARPE-19 human RPE cell lines after high glucose treatment and investigated its functions in high glucose- induced reactive oxygen species (ROS) generation and apoptotic responses. The downstream target gene that mediated the action of miR-383 was functionally characterized. It was found that high glucose induced a 2.4-fold increase in miR-383 abundance, compared to ARPE-19 cells treated with normal glucose. Overexpression of miR-383 inhibited cell viability and promoted apoptosis and ROS formation in ARPE-19 cells, which was coupled with deregulation of Bcl-2 and Bax. Peroxiredoxin 3 (PRDX3) expression was repressed by miR-383 in ARPE-19 cells. Restoration of PRDX3 counteracted miR-383-induced ROS generation and apoptosis, while silencing of PRDX3 phenocopied the detrimental effects of miR-383 on ARPE-19 cells. Delivery of anti-miR-383 inhibitors led to an increase of PRDX3 expression and prevented high glucose-elicited ROS formation and apoptosis in ARPE-19 cells. Overall, miR-383 upregulation accounts for high glucose-induced oxidative stress and apoptosis in RPE cells by repressing PRDX3 expression. Targeting miR-383 may have therapeutic potential in the treatment of diabetic retinopathy.