Abstract: Salvianolic acid B, rosmarinic acid and methyl lithospermate were successfully separated from the roots of Salvia miltiorrhiza Bunge by pH-zone-refining high speed countercurrent chromatography in one step.The separation was performed with a two-phase system composed of petroleum ether-ethyl acetate-butanol-water(2:3:6:9, the volume ratio).The lower-phase was used as the mobile phase with a flow rate of 2.0 mL/min, the apparatus rotated at 850 r/min and the effluents were detected at 254 nm.Salvianolic acid B(1 419.15 mg), rosmarinic acid(171.25 mg) and methyl lithospermate(52.34 mg) were obtained from 6 g of the crude sample in one step.Their purities determined by high performance liquid chromatography(HPLC)were 94.53%, 96.36% and 96.83%, respectively.The structures of the target compounds was identified by electrospray ionization mass spectrometry(ESI-MS), 1H nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR).