The determination of the chemical structure of an isolated vasodilatory molecule from Ludwigia octovalvis (Jacq.) P.H. Raven (Onagraceae) was undertaken. This molecule was isolated from n-butanolic extract using bioassay-guided isolation with preparative plate chromatographic techniques and its structure was determined using spectroscopy (UV, 1D- and 2D NMR, MS). The vasodilatory activity of the isolate was investigated on guinea pig isolated aorta. The results show 3 spots from n-butanolic extract with respective retention factor (Rf) of 0.14, 0.6 and 0.91. Pharmacological investigations showed that the isolated molecule with Rf 0.91 relaxes the isolated aorta pre-contracted by norepinephrine. Data from chemical shifts (δH) and spin-spin coupling constants (JHH) were compared with literature to confirm the isolate as rutin. This is the first report of rutin isolated from Ludwigia octovalvis.