Objective: Hyperglycemia is an important risk factor for the development of diabetic retinopathy. Recently we showed that high glucose activates NFAT in cerebral artery smooth muscle through local release of extracellular nucleotides, acting on P2Y receptors, leading to increased [Ca2+]i and calcineurin activation. NFAT activation promotes cell proliferation and the expression of inflammatory markers, such as IL-6, COX-2 and osteopontin, which are elevated in diabetic retinopathy. Here we investigate whether: 1) NFAT is activated by hyperglycemia in the wall of retinal microvessels (RM) and 2) inhibition of NFAT signaling may have a protective effect on the retina of diabetic mice. Results and methods: Using confocal immunofluorescence microscopy we show that NFATc3 is expressed in the endothelium of RM and readily activated by high glucose ex vivo and in vivo (intra peritoneal glucose tolerance test). This activation is inhibited by the NFAT blocker A- 285222 and by the ecto-nucleotidase apyrase. Chronic hyperglycemia in STZ-treated NFAT-luc mice leads to increased NFAT transcriptional activity in isolated RM. Interestingly, two weeks after the first STZ injection, levels of the potent anti-inflammatory cytokine IL-10 were decreased in retina and this was prevented by treatment with A-285222. Conclusion: Results suggest that NFAT inhibition may protect the retina of diabetic mice. Funding: Swedish Heart & Lung Foundations, Research Medical Council and RAC HSC-UNM 624195