This study examined the effect of chloroquine on vasodilation induced by levcromakalim in isolated endothelium-denuded rat aortas and clarified the underlying mechanisms. We examined the effects of chloroquine, hydroxychloroquine, lipid emulsion, reactive oxygen species (ROS) scavenger N-acetyl-ʟ-cysteine (NAC), and KATP channel inhibitor glibenclamide on levcromakaliminduced vasodilation. The effects of chloroquine, hydroxychloroquine, NAC, and levcromakalim on membrane hyperpolarization and ROS production were examined in aortic vascular smooth muscle cells (VSMCs). Chloroquine inhibited levcromakalim-induced vasodilation more than hydroxychloroquine. NAC attenuated chloroquine-mediated inhibition of levcromakalim-induced vasodilation, while lipid emulsion had no effect. Glibenclamide eliminated levcromakalim-induced vasodilation in aortas pretreated with chloroquine. Chloroquine and hydroxychloroquine inhibited levcromakalim-induced membrane hyperpolarization in VSMCs. Chloroquine and hydroxychloroquine both produced ROS, but chloroquine produced more. NAC inhibited chloroquine-induced ROS production in VSMCs. Collectively, these results suggest that, partially through ROS production, chloroquine inhibits levcromakalim-induced vasodilation. In addition, chloroquine-induced KATP channel-induced vasodilation impairment was not restored by lipid emulsion.