Six compounds including two n-butyrophenone isomers and two stibene isomers were obtained from Rheum tanguticum Maxim. Two n-butyrophenone isomers with a separation factor of 1.14 were successfully separated by recycling high-speed counter-current chromatography after ten cycles. Two stibene isomers were successfully separated by preparative high-performance liquid chromatography. High-performance liquid chromatography analysis showed that the purities of the compounds were all over 98%. These compounds were identified as lindleyin, isolindleyin, resveratrol-4'-O-(2″-O-galloyl)-glucopyranoside, resveratrol-4'-O-(6''-O-galloyl)-glucopyranoside, emodin 1-O-β-d-glucoside, and 3,5-dihydroxy-4'-methoxystilbene-3-O-β-d-glucopyranoside. The results indicated that recycling high-speed counter-current chromatography and preparative high-performance liquid chromatography could be effective combination for the preparation of bioactive compounds from Rheum tanguticum Maxim.