High-speed counter-current chromatography was used for separation and purification of natural pigment from the mycelia of Mariannaea puinosa.The tow-phase solvent system was ethyl acetate-n-butanol-methanol-water(10∶3∶3∶10).The upper phase was stationary phase and the lower phase was mobile phase.The flow-rate of the mobile phase was 2.0 mL/min and the rotation speed was 850 r/min.The effluent of the column was monitored with a UV detector at 340 nm.The purity of the pigment fraction was evaluated by high-performance liquid chromatography(HPLC).High resolution MS analysis revealed that the molecular formula of the compound was C26H32O9,which was possibly a new compound according to the searching results of Chapman & Hall natural products database.