Abstract: Due to the various bio- activities,Ergosterol peroxide presents great application value. The column chromatography is the most commonly methods used in separation of this compound. However,sample loss caused by irreversible adsorption is very uneconomical and biggest challenges in industrial application.In present work,an effective and rapid high- speed counter- current chromatography( HSCCC) method for isolating and purifying ergosterol peroxide from Xylaria Striata has been developed.After using HPTLC and HPLC to determinate the partition coefficient,the isolation efficiency of different solvent systems was observed.The result showed that the solvent system composed of n- hexane,ethyl acetate,ethanol and water( 3 ∶ 1 ∶ 2 ∶ 0.8,v ∶ v ∶ v ∶ v) was the best,the lower phase was used as the mobile phase and performed at a flow rate of 2 m L / min,while the apparatus rotated at 800 r / min,and detected at 220 nm.The prepared ergosterol peroxide was identified by13C-NMR and1H-NMR. Its purity was 96.05% analyzed by high performance liquid chromatography.And the antioxidant potential of ergosterol peroxide was also investigated by the method of DPPH radical scavenging activity. Results revealed that ergosterol peroxide showed weaker DPPH radical scavenging activity( 28.36%) at the concentration of 0.2 mg / m L compared with standard compound ascorbic acid( 93.41%).The dose dependent was not observed also explained its weak DPPH radical scavenging activity.The established method was quite simple,fast,and suitable for the large- scale isolation and separation of ergosterol peroxide from Xylaria Striata.