Background We investigated the role of calcium-activated potassium (KCa ) channel activity in human skeletal muscle microvascular function in the setting of cardiopulmonary bypass (CPB). Methods and Results Human skeletal muscle arterioles (80- to 180 μm in diameter) were dissected from tissue harvested before and after CPB. In vitro relaxation responses of precontracted arterioles in a pressurized no-flow state were examined in the presence of KCa channel activators/blockers and several other vasodilators. Post-CPB responses to the activator of intermediate (IKCa ) and small conductance (SKCa ) KCa channels, NS309, to the endothelium-dependent vasodilator adenosine 5′-diphosphate (ADP), and to substance P were reduced compared with pre-CPB responses ( P < .05), respectively, whereas responses to the activator of large conductance (BKCa ) KCa channels, NS1619, and to the endothelium-independent vasodilator, sodium nitroprusside (SNP) were unchanged. Endothelial denudation decreased NS309-induced relaxation and abolished that induced by ADP or substance P ( P < .05), but had no effect on relaxation induced by either NS1619 or SNP. Polypeptide levels of BKCa , IKCa , and SK3Ca were not altered post-CPB. Conclusion IK/SK-mediated relaxation is predominantly endothelium dependent, whereas BK-mediated relaxation seems to be largely independent of endothelial function in human skeletal muscle microvasculature. CPB-associated microvascular dysfunction likely arises in part from impaired function of endothelial SK and IK channels in the peripheral microvasculature.