In this study, we used the phenylephrine-precontracted aortic ring preparation to investigate the mechanisms underlying the vasorelaxant effect of linagliptin. We found that linagliptin induced vasorelaxation in a dose-dependent manner. The vasorelaxant effect of linagliptin was not changed by the removal of the endothelium, or by pre-treatment with a NO synthase inhibitor (L-NAME) or a small-conductance Ca2+-dependent K+ channel inhibitor (apamin). Moreover, administration of the adenylyl cyclase inhibitor Q122536, protein kinase A (PKA) inhibitor KT5720, guanylyl cyclase inhibitor ODQ, or protein kinase G (PKG) inhibitor KT5823 did not change the vasorelaxant effect of linagliptin. However, blocking of Rho-associated protein kinase by Y-27632 significantly reduced linagliptin-induced vasorelaxation. Involvement of vascular ion channel in the vasorelaxant effect of linagliptin was also investigated. Pre-treatment with the vascular K+ channel inhibitors Ba2+, 4-AP, paxilline, and glibenclamide did not affect linagliptin-induced vasorelaxation. Furthermore, the L-type Ca2+ channel inhibitor, nicardipine, and the SERCA pump inhibitor, cyclic piazonic acid, were not related with the vasorelaxant effect of linagliptin. From these findings, we concluded that linagliptin-induced vasorelaxation was mediated by the inhibition of Rho-associated kinase, but not with the endothelium, PKA or PKG-dependent signaling cascades, vascular K+ channels, Ca2+ channels, or intracellular Ca2+.