The vasodilatory effects of the meglitinide class of anti-diabetic drug, mitiglinide in phenylephrine (Phe)-pre-contracted aortic rings were investigated. Mitiglinide concentration-dependently induced vasodilation. Administration of the large-conductance Ca2+-dependent K+ (BKCa) channel inhibitor paxilline, inward rectifier K+ (Kir) channel inhibitor Ba2+, and ATP-dependent K+ (KATP) channel inhibitor glibencalmide did not affect the vasodilatory effect of mitiglinide. However, application of the voltage-dependent K+ (Kv) channel inhibitor 4-AP, effectively reduced mitiglinide-induced vasodilation. Although pre-treatment with the Ca2+ channel inhibitor nicardipine did not change the mitiglinide-induced vasodilation, pre-treatment with the SERCA pump inhibitor cyclopiazonic acid and thapsigargin reduced the vasodilatory effect of mitiglinide. In addition, the vasodilatory effect of mitiglinide was not changed by the blockers of adenylyl cyclase, guanylyl cyclase, protein kinase A, or protein kinase G. Removal of the endothelium and inhibition of endothelium-dependent vasodilatory mechanisms also did not affect the vasodilatory effect of mitiglinide. Based on these results, we suggested that mitiglinide induces vasodilation via activation of SERCA pump and Kv channels. However, the vasodilatory effects of mitiglinide did not related with other Ca2+ channels, K+ channels, PKA/PKG signaling pathways, or the endothelium.