An increase in O-linked N-acetylglucosamine (O-GlcNAc) protein modifications has been observerd in db/db mouse retinas. O-GlcNAc-modified proteins in the db/db mouse retina have been shown to be localized in the ganglion cell layer, the inner nuclear layer, the retina pigment epithelium (RPE) layer and the inner plexiform layer, in which hypoxia-inducible factor 1α (HIF1α) has also been shown to be localized. In the current study, we examined whether hypoxia increases O-GlcNAcylation in retinal vascular cells under high glucose conditions and whether HIF1α activation is consistent with the response to and activation of O-GlcNAcylation in retinal lesions in diabetic retinopathy. In addition, the effects of O-GlcNAcylation on the blood-retinal barrier were verified in vitro by the inhibition of O-GlcNAcylation. A time-dependent increase in the O-GlcNAcylation in bovine retinal vascular endothelial cells (BRVECs) was observed following incubation of the cells with high glucose medium (glucose 4.5 g/l) under hypoxic (1-3% O2) conditions. Hypoxia-induced BRVEC O-GlcNAcylation was not observed when the BRVECs were transfected with siRNA targeting O-GlcNAc transferase (OGT) or treated with alloxan (an OGT inhibitor) prior to exposure to high glucose. The increase in BRVEC O-GlcNAcylation induced by high glucose, as well as by thiamet G [an O-GlcNAcase (OGA) inhibitor] led to a reduction in occludin expression levels in vitro, which was prevented by treatment with OGT siRNA and alloxan. In conclusion, the current study demonstrates the relationship between O-GlcNAc glycosylation and hypoxia during diabetic retinopathy and that hyperglycemia induced O2 consumption activates HIF1α and O-GlcNAc modification protein in the same retinal layer. The reduced protein BRVEC O-GlcNAcylation levels exert protective effects on the blood-retinal barrier and thus represent a potential therapeutic target for the treatment of diabetic retinopathy.