The present study was undertaken to investigate the influence of cardamonin on vascular smooth muscle contractility and to determine the mechanism(s) involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Cardamonin significantly relaxed fluoride-, phenylephrine-, and phorbol ester-induced vascular contractions, suggesting that it has an anti-hypertensive effect on agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, cardamonin significantly inhibited the fluoride-induced increase in pMYPT1 level and phenylephrine-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. This study provides evidence that the relaxing effect of cardamonin on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activity.