Objective: Separation and purification of osthole by high-speed counter-current chromatography (HSCCC) from crude extract of Angelicae Pubescentis Radix. Method: Ultra high performance liquid (UPLC) was used to analyze and optimize the solvent system and the hexane-ethyl acetate-water-methanol (1.5: 2.5: 2.0: 1.5) was chosen as the two-phase solvent system

in which the upper phase was used as the stationary phase

while the lower phase was used as the mobile phase

the flow rate was set at 3.0 mL·min-1

the revolution speed was 850 r·min-1

and detected at 323 nm. The fraction was received and the solvent evaporated at 50 ℃ using a rotary evaporator

then the osthole was received and its purity was determined through UPLC

and its structure was identified by IR

ESI-MS

1H-NMR and 13C-NMR. Result: We got 6.6 mg of osthole one time and its purity was up to 97.1%. Conclusion: The high-speed counter-current chromatography is a simple and high-efficiency method and through which we can obtain osthole with high purity from crude extract of Angelicae Pubescentis Radix.