Ethanol is known to induce NO release and coronary vasorelaxation. Evidence suggests that K+ channels, especially a Ca2+-activated K+ channel (KCa), may regulate endothelial NO production. We aimed to investigate the ethanol effect on K+ currents in human coronary artery endothelial cells (HCAECs), identify the K+ channel type/subtype and signaling pathway involved, and demonstrate the relevance to ethanol-induced NO release. Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. NO production were measured using the fluorescent probe, 2,3-diaminonaphthalene. We found that ethanol significantly potentiated HCAEC current (maximal increase to 155.68 ± 18.93%, 20 mM ethanol, +80 mV; mean ± SEM, n = 9). Ethanol-induced current was significantly inhibited by blockers of IKCa or SKCa (intermediate- or small-conductance KCa), but not by blocking other K+ channels. When other known HCAEC channels were inhibited except IKCa, 20 mM ethanol significantly increased IKCa current to 198 ± 25.11% (n = 6), but it could not enhance SKCa current that was similarly isolated. Moreover, ethanol-induced NO release was prevented by blocking IKCa channel, adenosine A2A receptor (A2AR), Gs protein, or protein kinase A (PKA). This study was the first to demonstrate that acute ethanol exposure could activate endothelial IKCa channel, via A2AR-Gs-PKA signaling, leading to increased whole-cell current and NO release, which could be an important mechanism underlying ethanol-induced NO release and vasodilation.