Abstract: A preparative high-speed counter-current chromatography (HSCCC) was used to isolate and separate curculigoside and curculigoside B from the famous traditional Chinese medicinal herb Curculigo orchioides. Some parameters including two-phase solvent system, separation temperature,flow rate of the mobile phase and revolution speed of the apparatus were all investigated, and a successfully isolation and purification was achieved at the following conditions: the two-phase solvent system composed of ethyl acetate-ethanol-water at a volume ratio of 5:1:5(v/v/v) was selected and the lower phase of the system was used as the mobile phase at the flow rate of 2.0ml/min, and the isolation temperature and revolution speed were set at 30◦C and 800rpm.The isolation produced a total of 14.5 mg curculigoside B and 72.8 mg curculigoside with purities of 96.5% and 99.4% determined by high performance liquid chromatography (HPLC) from 300 mg crude extract after cleaning-up by D101 macroporous resin,which was necessary for the excellent purification. The recoveries of the two compounds were 91.6% and 92.5%, respectively, and the chemical structure identification was carried out by UV, MS and NMR. This separation method was more effective than some conventional techniques. TBE-300A高速逆流色譜儀/制備色譜儀 TBE300B+AKTA高速逆流色譜儀/離心分配色譜/萃取儀/制備色譜儀 TBE-1000A制備色譜儀/萃取儀/快速分離制備色譜儀 TBE-200V 高速逆流色譜儀 TBE-20A分析型高速逆流色譜儀/萃取儀/制備色譜儀