In this study, we explored vildagliptin-induced vasorelaxation and its related signaling mechanisms using phenylephrine induced pre-contracted rabbit aortic rings. Vildagliptin induced vasorelaxation in a dose-dependent fashion. Pre-treatment with the large-conductance Ca2+-dependent K+ channel inhibitor paxilline, ATP-dependent K+ channel inhibitor glibenclamide and inward rectifier K+ channel inhibitor Ba2+ did not change the vasorelaxant effects of vildagliptin. However, administration of the voltage-gated K+ (Kv) channel inhibitor 4-aminopyridine significantly decreased the vasorelaxant effects of vildagliptin. In addition, application of two SERCA inhibitors, cyclopiazonic acid or thapsigargin, effectively reduced the vasorelaxant effects of vildagliptin. These vasorelaxant effects were not changed by pretreatment with adenylyl cyclase, guanylyl cyclase, protein kinase A (PKA), or protein kinase G (PKG) inhibitors, or by elimination of the endothelium. Based on these results, we suggested that vildagliptin induced vasorelaxation via activation of the SERCA pump and Kv channels. However, PKA/PKG-related signaling pathways associated with vascular relaxation, other K+ channels, and the endothelium was not related in vildagliptin-induced vasorelaxation.