Irisin, an exercise-induced myokine, induces conversion of white into brown adipocytes, promoting mitochondrial biogenesis and energy expenditure. Irisin has a vascular protective effect on endothelial function in animals, including humans. Defects in irisin signaling pathways result in endothelial dysfunction in obesity and diabetes. However, the mechanisms underlying the effects of irisin on endothelial function have not been elucidated. Transient receptor potential vanilloid subtype 4 (TRPV4) channels are one of the most important Ca2+-permeable cation channels in vascular endothelial cells. In this study, we hypothesized that irisin may induce endothelium-dependent vasodilation by activating Ca2+ influx into endothelial cells via TRPV4 channels. In primary cultured rat mesenteric artery endothelial cells, irisin caused an increase in Ca2+i due to extracellular Ca2+ influx rather than release from Ca2+ stores. Moreover, irisin-induced increases in Ca2+i were completely abolished by a TRPV4 inhibitor. In addition, irisin induced endothelium-dependent vasodilation of rat mesenteric arteries. However, irisin had no effect on endothelium-independent vasodilation. Furthermore, irisin-induced vasodilation was fully abolished in the presence of a TRPV4 inhibitor, indicating the involvement of TRPV4 channels in endothelium-dependent vasodilation. This study provides the first evidence that irisin-induced endothelium-dependent vasodilation is related to the stimulation of extracellular Ca2+ influx via TRPV4 channels in rat mesenteric arteries. •Irisin caused an increase in Ca2+i due to extracellular Ca2+ influx rather than Ca2+ release in endothelial cells.•Pretreatment with TRPV4 channel inhibitor completely abolished irisin-induced increases in Ca2+i.•Irisin induced endothelium-dependent vasodilation, however, it had no effect on endothelium-independent vasodilation.•Pretreatment with TRPV4 channel inhibitor fully abrogated irisin-induced vasodilation.