Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) contribute to endothelium-dependent relaxation in small vessels such as mesenteric artery. Diabetes is known to impair endothelium-dependent relaxation, however, it is not established whether it affects either or both NO and EDHF. The aim of this study is to evaluate the mechanism of endothelium-dependent relaxation of mesenteric arteries from type 1 diabetic rats. Wire myography was employed to examine ACh-induced relaxation of mesenteric arteries. Superoxide levels, measured by L-012 and lucigenin-enhanced chemiluminescence, were significantly increased in diabetic mesenteric arteries compared to normal arteries. Diabetes significantly reduced the sensitivity to ACh (pEC ₅₀, diabetic, 6.77±0.14 vs normal, 7.74±0.09, n=10–11, p<0.0001) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA, 100 mM, Sigma-Aldrich, 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 compared to normal arteries (pEC ₅₀ diabetic, 6.68±0.12 vs normal, 7.08±0.11, n=11–12, p<0.05), indicating an impaired contribution of EDHF. Conversely, when the contribution of EDHF was inhibited with the combination of 1-[(2- chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34,1 mM, Sigma-Aldrich, USA), apamin (1 mM, Sigma-Aldrich, USA) and iberiotoxin (100 nM, Sigma-Aldrich, USA), the maximum relaxation (R_max) to ACh was significantly decreased in diabetic arteries (R _max diabetic 17±5 vs normal, 81±5, n=8–9, p<0.0001), suggesting that the contribution of NO was also impaired by diabetes. Western blot analysis also demonstrated eNOS uncoupling in diabetic mesenteric arteries. Taken together, this study demonstrated that endothelium- dependent relaxation was impaired at 10 weeks of diabetes due to a reduced contribution of both NO and EDHF.