Prenatal testosterone (T) exposure impacts postnatal cardiovascular function, leading to increases in blood pressure with associated decreased endothelium-dependent vascular relaxation in adult females. Endothelial function in males is not known. Furthermore, which of the endothelial pathways contributes to endothelial dysfunction and if there exists sex differences are not known. The objective of this study was to characterize the relative contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the impaired endothelium-dependent vasodilation in prenatal T-exposed adult males and females. Offspring of pregnant rats treated with T propionate or its vehicle were examined. Telemetric blood pressure levels and endothelium-dependent vascular reactivity were assessed with wire myography. Levels of nitric oxide synthase (NOS3) and Kcnn3 and Kcnn4 channel expression were examined in mesenteric arteries. Mean arterial pressure was significantly higher in T males and females than in controls. Endothelium-dependent acetylcholine relaxation was significantly lower in both T males and females. EDHF-mediated relaxation was specifically blunted in T males (Emax = 48.64% ± 3.73%) compared to that in control males (Emax = 81.71% ± 3.18%); however, NO-mediated relaxation was specifically impaired in T females (Emax = 36.01% ± 4.29%) compared with that in control females (Emax = 54.56% ± 6.37%). Relaxation to sodium nitroprusside and levcromakalim were unaffected with T-treatment. NOS3 protein was decreased in T females but not in T males. Kcnn3 expression was decreased in both T males and females compared to controls. These findings suggest that prenatal T leads to an increase in blood pressure in the adult offspring, associated with blunting of endothelial cell-associated relaxation and that the effects are sex-specific: EDHF-related in males and NO-related in females.