Maytansinoids are a family of 19-membered macrocyclic lactams with extraordinary cytotoxic and antineoplastic activities, produced by Actinosynnema pretiosum, mosses, and three closely related plant families (Celastraceae, Rhamnaceae, Euphorbiaceae). By cultivating A. pretiosum on YMG agar media (yeast extract 4 g/liter, malt extract 10 g/liter, glucose 4 g/liter), we isolated a novel ansamitocin amide N-glycoside, N-demethyl-N-β-D-glucopyranosyl ansamitocin P-2 (named ansamitocinoside P-2, compound 1), through column chromatography over reversed-phase C18 silica gel and Sephadex LH-20. The structure elucidation of ansamitocinoside P-2 was unambiguously carried out on the basis of HRFABMS, and 1D and 2D NMR data. Compound 1 was determined to have the molecular formula C36H48ClN2O14. The ansamitocin moiety (aglycone) had the same stereochemistry as ansamitocins. The sugar moiety was identified as β-D-glucopyranosyl, and glycosylation occurred at the amide nitrogen of C-18. Therefore, compound 1 is a novel amide N-glycoside of ansamitocins.