Background and purpose KCNQ-encoded KV7 channels are expressed within vascular smooth muscle cells (VSMCs) and are key regulators of vascular reactivity, regulating resting tone and as functional targets of endogenous responses. Endothelial cells (ECs) form a paracrine signaling platform that line all blood vessels and regulate tone, but little is known of KV7 channels in vascular ECs. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental approach In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key results KCNQ transcript was identified in EC marker expressing cells using a reductive approach. KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133 and BaCl also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusions and implications In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium.