Hypercholesterolemic patients display reduced coronary flow reserve in response to adenosine infusion. We previously reported that voltage-dependent K⁺ (Kv) channels contribute to adenosine-mediated relaxation of coronary arterioles isolated from male miniature swine. For this study, we hypothesized that hypercholesterolemia attenuates Kv channel contribution to adenosine-induced vasodilatation. Pigs were randomly assigned to a control or high fat/high cholesterol diet for 20-24 weeks, and then sacrificed. After completion of the experimental treatment, arterioles (~150 µm luminal diameter) were isolated from the left-ventricular free wall near the apical region of the heart, cannulated and pressurized at 40 mmHg. Adenosine-mediated relaxation was significantly attenuated in both endothelium-intact and -denuded arterioles from hypercholesterolemic compared with control animals. The classic Kv channel blocker, 4-aminopyridine (4-AP; 1 mM), significantly attenuated adenosine-mediated relaxation in arterioles isolated from control but not hypercholesterolemic animals. Furthermore, the nonselective K⁺ channel blocker, tetraethylammonium (TEA; 1 mM) significantly attenuated adenosine-mediated relaxation in arterioles from control but not hypercholesterolemic animals. In additional experiments, coronary arteriolar smooth muscle cells were isolated and whole-cell Kv currents measured. Kv currents were significantly reduced (~15 %) in smooth muscle cells from hypercholesterolemic compared with control animals. Furthermore, Kv current sensitive to low concentrations of TEA was reduced (~45 %) in smooth muscle cells from hypercholesterolemic compared with control animals. Our data indicate that hypercholesterolemia abolishes Kv channel contribution to adenosine-mediated relaxation in coronary arterioles, which may be attributable to a reduced contribution of TEA-sensitive Kv channels in smooth muscle of hypercholesterolemic animals.