Background and Purpose Nuciferine, a constituent of lotus leaf, is an aromatic ring-containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanism Experimental Approach Nuciferine-induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging. Key Results Nuciferine induced relaxations in arterial segments pre-contracted by KCl or phenylephrine. Nuciferine-elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L-NAME or the NO-sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca influx. Under endothelium-free conditions, nuciferine attenuated CaCl-induced contraction in Ca-free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl. Nuciferine also suppressed Ca influx in Ca-free K-containing solution in VSMCs. Conclusions and Implications Nuciferine has a vasorelaxant effect via both endothelium-dependent and -independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction. Linked Articles This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery.