A novel polyhydroxylated C(29)-sterol, 25xi-methyl-22-homo-5alpha-cholest-7,22-diene-3beta,6beta,9alpha-triol, designated globosterol (1), together with one known tetrahydroxylated ergosterol (22E, 24R)-ergosta-7,22-diene-3beta,5alpha,6beta,9alpha-tetraol (2) has been isolated from the cultures of an endophytic fungus, Chaetomium globosum ZY-22 originated from the plant Ginkgo biloba. The structures and relative configurations of 1 and 2 were established on the basis of extensive spectroscopic analyses including 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC, and NOESY) experiments and comparison with the literature. Globosterol (1) possesses an unprecedented 25-methyl Delta(22)-C(10)-side chain and Delta(7)-3beta,6beta,9alpha-hydroxy-steroid nucleus, which represents the first example for C(29)-steroids of the group.