ATP-sensitive K⁺ channels (K_ATP) contribute to vasomotor regulation in some species. It is not fully understood the extent to which K_ATP participate in regulating vasomotor tone under physiological and pathophysiological conditions in the human heart. Arterioles dissected from right atrial appendage were studied with video microscopy, membrane potential recordings, reverse transcription–polymerase chain reaction, and immunohistochemistry. Hypoxia produced endothelium-independent vasodilation and membrane hyperpolarization of vascular smooth muscle cells, both of which were attenuated by glibenclamide. Aprikalim, a selective K_ATP opener, also induced a potent endothelium-independent and glibenclamide-sensitive vasodilation with membrane hyperpolarization. Reverse transcription–polymerase chain reaction detected mRNA expression for K_ATP subunits, and immunohistochemistry confirmed the localization of the inwardly rectifying Kir6.1 protein in the vasculature. In patients with type 1 or type 2 diabetes mellitus (DM), vasodilation was reduced to both aprikalim (maximum dilation, DM(+) 90±2% versus DM(−) 96±1%, P<0.05) and hypoxia (maximum dilation, DM(+) 56±8% versus DM(−) 85±5%, P<0.01) but was not altered to sodium nitroprusside or bradykinin. Baseline myogenic tone and resting membrane potential were not affected by DM. We conclude that DM impairs human coronary arteriolar dilation to K_ATP opening, leading to reduced dilation to hypoxia. This reduction in K_ATP function could contribute to the greater cardiovascular mortality and morbidity in DM.