Key points KCNE4 alters the biophysical properties and cellular localisation of Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co‐localises with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarised membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle. Abstract The KCNE ancillary subunits (KCNE1‐5) significantly alter Kv7 channel expression and function; however, their role in the vasculature has yet to be determined. The aim of this study was to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co‐expression of KCNE4 increased membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. QPCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed KCNE4 co‐localised with Kv7.4 in mesenteric artery myocytes. Morpholino‐induced knockdown of KCNE4 depolarised mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine (EC50 decreased from 5.7 ± 0.63 μm to 1.6 ± 0.23 μm), which coincided with the effects of Kv7 modulators, being attenuated. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarised membrane potential and an augmented response to vasoconstrictors. This study is the first to ascertain an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle. This article is protected by copyright. All rights reserved