A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of costunolide and dehydrocostuslactone from the Chinese medicinal plant Aucklandia lappa Decne (Muxiang in Chinese) was successfully established by using light petroleum-methanol-water (5:6.5:3.5, v/v/v) as the two-phase solvent system. The upper phase of light petroleum-methanol-water (5:6.5:3.5, v/v/v) was used as the stationary phase of HSCCC. 35.7 mg of costunolide and 43.6 mg of dehydrocostuslactone with the purity of 100% and 99.6%, respectively, were separated successfully in one-step separation from 110 mg of crude sample from Aucklandia lappa Decne. The structures of costunolide and dehydrocostuslactone were identified by 1H NMR and 13C NMR.