Abstract: Piperine, a major plant alkaloid presenting in pepper, was successfully separated and purified from the crude extract of Piper nigrum Linn. by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether (60-90℃)-ethyl acetate-methanol-water (1:0.8:1:0.8, volume ratio). The upper phase was used as stationary phase, while the lower phase was used as mobile phase. From 300 mg of crude extract, 65.2 mg of piperine was obtained at 99.1% purity in one-step separation. The structure of the target compound was identified by EI-MS, 1H NMR and 13C NMR.