Little is known about interaction of sex and diabetes in vasculature. Our study investigates the effects of type 2 diabetes on endothelium-dependent and -independent relaxations. Also, if there are sex-based changes in relative contributions of endothelium derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries (MA) from ZDF rats. Relaxation responses to acetylcholine (ACh) in MA pre-contracted with phenylephrine (PE) were obtained before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L-NAME (nitric oxide synthase inhibitor) or barium chloride (Kir blocker) plus ouabain (Na+-K+-ATPase inhibitor). Vascular responses to sodium nitroprusside (SNP) were also measured in MA. ACh-induced relaxations were significantly impaired in MA of diabetic rats, regardless of sex. In diabetic females, the relative importance of endothelium derived hyperpolarizing factor (EDHF) in relaxation to ACh was reduced, while in diabetic males, role of nitric oxide (NO) was reduced. Interestingly, relaxation to SNP was enhanced in diabetic animals, irrespective of sex. In summary, the relative importance of NO and EDHF in regulating vascular tone of rat MA is altered in type 2 diabetes with respect to sex. Furthermore, increased smooth muscle sensitivity to NO may be an attempt to compensate for impaired endothelial function in both diabetic male and female rats. Grant Funding Source: Supported by NIDCR.