-Cyperone was separated and purified for the first time from essential oil of the traditional Chinese medicinal plant Cyperus rotundus L. by high-speed counter-current chromatography (HSCCC). Essential oil was obtained by extraction with supercritical carbon dioxide under the pressure of 20MPa and temperature of 40C. The separation was performed in one step with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.2:1.1:0.2, v/v), in which the lower phase was used as the mobile phase at a flow-rate of 2.0ml/min in the head-to-tail elution mode. A total of 60mg -cyperone at 98.8% purity was yielded from 0.9g essential oil as determined by HPLC analysis. The structure of the target compound was performed by electron impact ionization mass spectrometry (EI-MS) and further conformed by comparison with an authentic sample (National Institute of the Control of Pharmaceutical and Biological Products, Beijing, China).