Abstract: Objective:Study on secondary metabolites from endophytic fungus AR-17-S03 isolated from the roots of Astragalus membranaceus in summer. Methods:Endophyic fungus was isolated from Astragalus membranaceus by the method of histology. The fungus was identified by morphological method combined with molecular biology method. The compounds were isolated by column chromatography,pTLC,Sephadex LH-20 and pre-HPLC. And they were identified by the spectral analysis of mass spectrometry and nuclear magnetic resonance. Results:Endophytic fungi AR-17-S03 isolated from roots of Astragalus membranaceus were identified as Aspergillus niger. Nine sterols were obtained and identified as ergosterol,ergosterol peroxide,cholesterol,cerevistero,23-methylergosta-7,22-diene-3β,5α,9α-triol,5α,8α-epidioxy-ergosta-6,9(11),22-trien-3β-ol,5α,8α-epidioxy-23-methylergosta-6,22-diene-3β-ol,ergosta-4,6,8(14),22-tetraen-3-one and 3β,5α-dihydroxy-ergosta-7,22-diene-6-one. Conclusion:23-methylergosta-7,22-diene-3β,5α,9α-triol,5α,8α-epidioxy-23-methylergosta-6,22-diene-3β-ol and ergosta-4,6,8(14),22-tetraen-3-one were obtained from the endophytic fungus of Astragalus membranaceus for the first time.