1. Resibufogenin (1), a major bufadienolide of Chinese medicine Chan Su, had a wide range of pharmacological activities. In present work, the metabolism of 1 in male Sprague-Dawley rats was investigated by identifying the metabolites of resibufogenin excreted in rat bile. 2. Following an oral dose of 60 mg/kg resibufagenin, nine metabolites were isolated from bile of rats, and their structures were identified as 3-keto- resibufogenin (2), 3-epi-resibufogenin (3), 5β-hydroxy-3-epi-resibufogenin (4), 1α, 5β-dihydroxy-3-epi-resibufogenin (5), 3α, 5β, 14α, 15β-tetrahydroxyl-bufa- 20, 22-dienolide (6), 3α, 14α, 15β-trihydroxy-bufa-20, 22-dienolide (7), 3-epi- 5β-hydroxy-bufalin (8), 12α, 16β-dihydroxy-3-epi-resibufogenin (9), and 5β, 16β-dihydroxy-3-epi-resibufogenin (10), respectively, on the basis of widely spectroscopic methods including 2D-NMR technology. It is first time to describe the metabolites of 1 in vivo, and metabolites 5-7 and 9-10 are novel. 3. On the basis of these identified metabolites, a possible metabolism pathway for 1 in rats has been proposed. This is the first systematic study on the phase I metabolites of resibufogenin.