Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A 2 (PLA 2 ) in diabetic retinopathy using an in vitro blood–retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA 2 activity, VEGF and PGE 2 levels and cell proliferation were measured, with or without PLA 2 inhibition. Diabetes was induced in rats by STZ injection and PLA 2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA 2 activity, PGE 2 and VEGF in EC/PC co-cultures; inhibition of PLA 2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE 2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA 2 . PLA 2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA 2 inhibitor. In conclusion, the present findings indicate that PLA 2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA 2 may be an interesting target in managing diabetic retinopathy.