Background Small conductance calcium‐activated potassium (SK) channels are largely responsible for endothelium‐dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes‐induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. Methods and Results SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12‐myristate 13‐acetate, PKC inhibitors, or endothelial PKC _α /PKC _β knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKC _β inhibitor LY333531. NADH (30–300 μmol/L) or PKC activator phorbol 12‐myristate 13‐acetate (30–300 nmol/L) reduced endothelial SK current density, whereas the selective PKC _ᵦ inhibitor LY333531 significantly reversed the NADH‐induced SK channel inhibition. PKC _β small interfering RNA, but not PKC _α small interfering RNA, significantly prevented the NADH‐ and phorbol 12‐myristate 13‐acetate–induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKC _β expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKC _β inhibitor LY333531. Conclusions NADH‐induced inhibition of endothelial SK channel function is mediated via PKC _β . These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.