Previous studies demonstrated that eupatorin content in Orthosiphon stamineus fractions correlated with their vasorelaxation activity. Even with previous studies, there is still very little information on the vasorelaxation effect of eupatorin, and not many scientific studies had been carried out. Therefore, the present study was designed to investigate the vasorelaxation activity and mechanism of action of eupatorin. The vasorelaxation activity and the underlying mechanisms of eupatorin was evaluated on thoracic aortic rings isolated from Sprague Dawley rats. Eupatorin caused the relaxation of aortic rings pre-contracted with phenylephrine with and without endothelium (pD2=6.66±0.13, EMAX=99.72±6.39%; pD2=6.10±0.22, EMAX=65.78±8.01%), and also the relaxation of endothelium-intact aortic rings pre-contracted with potassium chloride (pD2=6.20±0.30, EMAX=71.89±12.25%). In the presence of Nω-nitro-l-arginine methyl ester (pD2<4.60, EMAX=24.91±6.39%), methylene blue (pD2=6.05±0.38, EMAX=66.79±9.69%), ODQ (pD25.84±0.32, EMAX=60.47±9.6%), indomethacin (pD2=6.27±0.21, EMAX=76.03±9.45%), tetraethylammonium (pD2=6.09±0.35, EMAX=69.35±11.31%), 4-aminopyridine (pD2=6.34±0.12, EMAX=76±6.1%), barium chloride (pD2=6.47±0.14, EMAX=79.61±10.02%), atropine (pD2=6.36±0.29, EMAX=86.47±12.95%) and propranolol (pD2=6.49±0.26, EMAX=83.2±12.01%), relaxation stimulated by eupatorin was significantly reduced. Eupatorin was also found to be active in reducing Ca2+ release from sarcoplasmic reticulum and in blocking calcium channels. The present study demonstrates the vasorelaxation effect of eupatorin involving NO/sGC/cGMP and indomethacin pathways, calcium and potassium channels, and muscarinic and beta-adrenergic receptors.