It has recently been reported that O-linked β-N-acetyl glucosamine (O-GlcNAc) modification (a simple intracellular serine (Ser)/threonine (Thr)-linked monosaccharide) in human retinal microvascular endothelial cells (HRECs) is related to diabetic retinopathy (DR). During O-GlcNAcylation, O-GlcNAc is added to Ser and Thr residues. As the generation of reactive oxygen species (ROS) is one of the characteristics of advanced glycation end product (AGE) injury, and the most important key pathogenic factor of DR, in the present study, we aimed to investigate the association between O-GlcNAcylation and ROS generation in order to ascertain whether O-GlcNAcylation mitigates cellular injury through the generation of ROS. For this purpose, HRECs were divided into 4 groups as follows: HRECs treated with normal glucose (5 mM), HRECs treated with glyoxal (500 µM), glyoxal-treated HRECs also treated with 200 µM PUGNAc, and glyoxal-treated HRECs infected with O-GlcNAc transferase (OGT) siRNA. We detected increased O-GlcNAc levels and increased ROS production in the glyoxal-treated HRECs. The cellular redox status was determined by cellular ROS staining and by measuring the expression levels of the antioxidant genes, superoxide dismutase (SOD) and glutathione peroxidase (GPX). While the augmentation of O-GlcNAcylation following treatment with PUGNAc significantly attenuated the production of ROS (p<0.01) and increased the expression levels of SOD and GPX, the reduction of O-GlcNAcylation following infection with OGT siRNA, exacerbated the production of ROS (p<0.01) and decreased the expression of antioxidant genes. The effects of O-GlcNAcylation on the viability of HRECs were significant (p<0.01), particularly in the hydrogen peroxide (H2O2)-treated HRECs. Treatment with PUGNAc reduced glyoxal-induced cell apoptosis and transfection with OGT siRNA increased HREC apoptosis; these results were confirmed by flow cytometry and by the assessment of mitochondrial membrane potential. The augmentation of O-GlcNAcylation exerted cytoprotective effects on the HRECs by reducing the generation of ROS, increasing the expression of antioxidant genes, preventing the dissipation of mitochondrial membrane potential and preventing HREC apoptosis. Therefore, it can be concluded that O-GlcNAcylation plays a role in the early developmental process of DR.