3', 4'-dihydroxyflavonol (DiOHF) prevents diabetes-induced endothelial dysfunction in aorta; however, it is unclear whether it is vasoprotective in the microvasculature. The aim of this study was to evaluate the effect of treating diabetic rats with DiOHF (7 days, 1mg/kg per day, s.c) on mesenteric artery function. Superoxide levels, determined by lucigenin-enhanced chemiluminescence, were significantly increased in diabetic mesenteric arteries but treatment with DiOHF reversed that effect. ACh-induced relaxation of mesenteric arteries was assessed using wire myography. Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC50 diabetic, 6.86±0.12 vs diabetic+DiOHF, 7.49±0.13, n=11, p<0.05) in mesenteric arteries. When the contribution of nitric oxide (NO) to relaxation was eliminated by N-nitro-L-arginine (L-NNA, 100 mM, Sigma, USA) and 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, 10 mM, Caymen, USA), the sensitivity to ACh was significantly decreased in the diabetic arteries (pEC50 diabetic, 6.63±0.15 vs normal, 7.14±0.12, n=12, p<0.05), and treatment with DiOHF had no effect (pEC50 diabetic, 6.85±0.12). Thus, DiOHF did not affect the contribution of endothelium-derived hyperpolarizing factor (EDHF) to relaxation. When the contribution of EDHF was inhibited with the potassium channel blockers, 1-[(2-chlorophenyl)(diphenyl)methyl]-1H- pyrazole (TRAM-34,1 mM, Sigma, USA), apamin (1 mM, Sigma, USA) and iberiotoxin (100 nM, Sigma, USA), the maximum relaxation (Rmax) to ACh was significantly decreased in diabetic arteries (Rmax diabetic 31±9 vs normal, 68±10, n=8–9, p<0.05), suggesting that diabetes impaired NO activity but DiOHF treatment prevented that effect (Rmax diabetic+DiOHF 69±6, n=11). Treatment with DiOHF in normal rats had no effect on superoxide levels or endothelial function. DiOHF treatment prevents endothelial dysfunction by preserving NO activity.