Plumbagin, a hydroxy 1,4-naphthoquinone compound from plant metabolites, exhibits anticancer, antibacterial, and antifungal activities via modulating various signaling molecules. However, its effects on vascular functions are rarely studied except in pulmonary and coronary arteries where NADPH oxidase (NOX) inhibition was suggested as a mechanism. Here we investigate the effects of plumbagin on the contractility of skeletal artery (deep femoral artery, DFA), mesenteric artery (MA) and renal artery (RA) in rats. Although plumbagin alone had no effect on the isometric tone of DFA, 1 μM phenylephrine (PhE)-induced partial contraction was largely augmented by plumbagin (ΔTPlum, 125% of 80 mM KCl-induced contraction at 1 μM). With relatively higher concentrations (>5 μM), plumbagin induced a transient contraction followed by tonic relaxation of DFA. Similar biphasic augmentation of the PhE-induced contraction was observed in MA and RA. VAS2870 and GKT137831, specific NOX4 inhibitors, neither mimicked nor inhibited ΔTPlum in DFA. Also, pretreatment with tiron or catalase did not affect ΔTPlum of DFA. Under the inhibition of PhE-contraction with L-type Ca2+ channel blocker (nifedipine, 1 μM), plumbagin still induced tonic contraction, suggesting Ca2+-sensitization mechanism of smooth muscle. Although ΔTPlum was consistently observed under pretreatment with Rho A-kinase inhibitor (Y27632, 1 μM), a PKC inhibitor (GF 109203X, 10 μM) largely suppressed ΔTPlum. Taken together, it is suggested that plumbagin facilitates the PKC activation in the presence of vasoactive agonists in skeletal arteries. The biphasic contractile effects on the systemic arteries should be considered in the pharmacological studies of plumbagin and 1,4-naphthoquinones.