Background <p><i>Ginkgo biloba</i> extract and its active component bilobalide induce endothelium-dependent vasodilation in coronary and other vascular beds. This effect is associated with increased Ca²⁺-activated K⁺ channel activity in endothelial cells. We aimed to identify ion channels targeted by bilobalide in human coronary artery endothelial cells (HCAECs) and to explore the underlying mechanisms.</p> Methods <p>Whole-cell patch-clamp recordings were performed on cultured HCAECs. Epoxyeicosatrienoic acids (EETs) and prostacyclin were quantified with enzyme-linked immunosorbent assays; nitric oxide and hydrogen peroxide were assessed with commercial kits.</p> Results <p>Bilobalide dose-dependently increased whole-cell current through activation of the small-conductance Ca²⁺-activated K⁺ (SK_Ca) channel, but not through Cl⁻, transient receptor potential, or other K⁺ channels. The current increase persisted when all channels except SK_Ca were blocked and was abolished by apamin, a selective SK_Ca blocker; additionally, apamin prevented bilobalide-stimulated current. Bilobalide also induced HCAEC hyperpolarization, which was inhibited by apamin. The bilobalide-induced current increase was blocked by antagonists of 5-hydroxytryptamine (5-HT)_1/2 and 5-HT_2B receptors, phospholipase C, and protein kinase C, but not by inhibitors of 5-HT_1B receptors, cytochrome P450, or G_i/o proteins. Furthermore, bilobalide stimulated EET production but not nitric oxide, prostacyclin, or hydrogen peroxide production; this effect was also apamin-sensitive.</p> Conclusions <p>Bilobalide increases HCAEC current and induces hyperpolarization by activating SK_Ca via the 5-HT_2B receptor–phospholipase C–protein kinase C signaling pathway, leading to EET generation. These findings provide insights into bilobalide’s endothelial mechanisms and highlight its potential as an alternative therapy for coronary artery disease.</p>