Accumulating data, including those from our laboratory, have shown that the opening of ATP‐sensitive potassium channels (K_ATP) plays a protective role in pulmonary vascular diseases (PVD). As maintainers of the endothelial framework, endothelial colony‐forming cells (ECFCs) are considered excellent candidates for vascular regeneration in cases of PVD. Although K_ATP openers (KCOs) have been demonstrated to have beneficial effects on endothelial cells, the impact of K_ATP on ECFC function remains unclear. Herein, this study investigated whether there is a distribution of K_ATP in ECFCs and what role K_ATP play in ECFC modulation. By human ECFCs isolated from adult peripheral blood, K_ATP were confirmed for the first time to express in ECFCs, comprised subunits of Kir (Kir6.1, Kir6.2) and SUR2b. KCOs such as the classical agent nicorandil (Nico) and the novel agent iptakalim (Ipt) notably improved the function of ECFCs, promoting cell proliferation, migration and angiogenesis, which were abolished by a non‐selective K_ATP blocker glibenclamide (Gli). To determine the underlying mechanisms, we investigated the impacts of KCOs on CaMKII, Akt and endothelial nitric oxide synthase pathways. Enhanced levels were detected by western blotting, which were abrogated by Gli. This suggested an involvement of Ca²⁺ signalling in the regulation of ECFCs by K_ATP. Our findings demonstrated for the first time that there is a distribution of K_ATP in ECFCs and K_ATP play a vital role in ECFC function. The present work highlighted a novel profile of K_ATP as a potential target for ECFC modulation, which may hold the key to the treatment of PVD.