Hyperglycemia is associated with impaired endothelium-dependent dilation that is due to quenching of NO by superoxide (O₂^{· −}). In small coronary arteries (CAs), dilation depends more on smooth muscle hyperpolarization, such as that mediated by voltage-gated K⁺ (Kv) channels. We determined whether high glucose enhances O₂^·− production and reduces microvascular Kv channel current and functional responses. CAs from Sprague-Dawley rats were incubated 24 hours in medium containing either normal glucose (NG, 5.5 mmol/L d-glucose), high glucose (HG, 23 mmol/L d-glucose), or l-glucose (LG, 5.5 mmol/L d-glucose and 17 mmol/L l-glucose). O₂^·− production was increased in HG arteries. Whole-cell patch clamping showed a reduction of 4-aminopyridine (4-AP)–sensitive current (Kv current) from smooth muscle cells of HG CAs versus NG CAs or versus LG CAs (peak density was 9.95±5.3 pA/pF for HG versus 27.8±6.8 pA/pF for NG and 28.5±5.2 pA/pF for LG; P<0.05). O₂^·− generation (xanthine+xanthine oxidase) decreased K⁺ current density, with no further reduction by 4-AP. Partial restoration was observed with superoxide dismutase and catalase. Constriction to 3 mmol/L 4-AP was reduced in vessels exposed to HG (13±5%, P<0.05) versus NG (30±7%) or LG (34±4%). Responses to KCl and nifedipine were not different among groups. Superoxide dismutase and catalase increased contraction to 4-AP in HG CAs. This is the first direct evidence that exposure of CAs to HG impairs Kv channel activity. We speculate that this O₂^·−-induced impairment may reduce vasodilator responsiveness in the coronary circulation of subjects with coronary disease or its risk factors.