In the course of our screening for specific apoptosis inducers in transformed cells, we isolated a novel 20-membered macrolide, apoptolidin (1), from Nocardiopsis sp. 1 induced apoptotic cell death in rat glia cells transformed with the adenovirus E1A oncogene (IC_<50> 11ng/ml) but not in normal glia cells or normal fibroblasts (IC_<50>>100μg/ml). The molecular formula of 1 was determined to be C_<58>H_<96>O_<21> from high-resolution FAB-MS. COSY and HMBC experiments generated a polyketide chain (2) and three hexoses (3〜5) as the partial structures. Methanolysis of 1 followed by hydrolysis yielded D-oleandrose (5), L-olivomycose (4), and a novel sugar, 6-deoxy-4-O-methyl-L-glucose (3). ^1-^<13>C long-range correlations constructed a 20-membered macrolide ring, a 6-membered hemiketal ring and three glycosidic linkages. High-field carbon shifts for the allylic methyls and a large vicinal coupling constant indicated all (E) configurations for the five olefinic bonds. A clue to the absolute stereochemistry of the macrolide ring was found between the 6-deoxy-4-O-methyl-L-glucose moiety and the vicinal ring methines (C-8 and C-9). NOEs including the most important one observed between 3-H and 17-H identified the absolute configurations of all the asymmetrical carbons. The stereochemistry of 1 thus obtained was confirmed by distance analysis in dihedral angle space (DADAS) using a JEOL MolSkop system.