To study the chemical constituents of Inonotus obliquus and their cytotoxicity and cholinesterase inhibitory activity.With various chromatographic methods,twelve compounds were isolated from the ethyl acetate fraction of I. obliquus. Their structures were identified on the basis of spectroscopic data analysis as monogynol A (1),betulinic acid (2),betulin (3),lanosterol (4),inoterpene D (5),inonotsutriol C (6),(3β,5α,6β,22E)-6-methoxyergosta-7,22-diene-3,5-diol (7),ergosterol peroxide (8),4-hydroxy-3-methoxybenzoic acid (9),2-(3,4-dihydroxyphenyl)-1,3-benzodioxole-5-carboxaldehyde (10),2,3-dihydroxy-1-4-hydroxy-3,5-dimethoxyphenyl-1-acetone (11),3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl) propan-1-one (12).Compounds 1,10 and 11 were isolated from the I. obliquus for the first time.The bioassay results showed that compounds 2,3,4,and 6 displayed butyrylcholinesterase inhibitory activity; Compounds 2 and 3 showed acetylcholinesterase inhibitory activity;compounds 1,5,and 6 exhibited moderate cytotoxic activity.