Hydrogen peroxide (H₂O₂) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H₂O₂ depending on the vascular bed and experimental conditions, this study was performed to assess whether H₂O₂ acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H₂O₂ elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H₂O₂ was reversed by treatment with dithiothreitol. H₂O₂-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca²⁺-activated K⁺ channels and inward rectifier K⁺ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H₂O₂ increased Kv currents in a concentration-dependent manner. H₂O₂ speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H₂O₂-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H₂O₂. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H₂O₂ to stimulate Kv channels. Not only was the H₂O₂ stimulatory effect much weaker, but the inhibitory effect of H₂O₂ was unmasked. These data suggest that H₂O₂ activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H₂O₂.