High-speed counter-current chromatography was successfully used for the first time for the preparative separation and purification of paeoniflorin from the Chinese medicinal plant Paeonia lactiflora Pall. using a two-phase solvent system composed of n-butanol-ethyl acetate-water (1:4:5, v/v) in a single run. From 160 mg of the crude sample containing 22.0% paeoniflorin, 33.2 mg of paeoniflorin was yielded at 98.2% purity as determined by HPLC analysis. The recovery of paeoniflorin was 94.3%.