In this study, daphnetin and its major metabolites in the intestinal wall of rats were identified by liquid chromatography and quatrupole-time of flight mass spectrometry.  Perfusion fluid of duodenum, jejunum, ileum and colon were collected separately for 2 hours from the rat intestine following perfusion with daphnetin.  The metabolites of daphnetin in the perfusion fluid of different intestine segments were analyzed by the liquid chromatography and quatrupole-time of flight mass spectrometry.  It is shown that the parent drug daphnetin and four metabolites were found in the perfusion fluid of duodenum, jejunum and ileum.  However, no metabolites were found in the colon.  Among the four metabolites, two daphnetin sulfates (m/z 257) were first discovered as the phase II metabolites of daphnetin in rats, which revealed a new way of daphnetin metabolism in rats.