A chemical investigation of the roots of Cynanchum stauntonii has resulted in the characterization of a new hydroperoxide with a 13,14 : 14,15-disecopregnane-type skeleton, named stauntonine (1), together with three related compounds, anhydrohirundigenin (2), anhydrohirundigenin monothevetoside (3), and glaucogenin-C mono- D-thevetoside (4). Their structures were established by spectroscopic methods, including X-ray crystallographic diffraction analysis of stauntonine that confirmed its relative stereochemistry. The compound 1 showed dose-dependent relaxation on aortic rings with endothelium contracted by phenylepherine or KCl.