AIM:To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine. METHODS:Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h postincubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 × from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin (100 mg/kg) was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes. RESULTS:Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-glucocoumarin (M1), 6-hydroxy-7-sulf-coumarin (M2), 6, 7-digluco- coumarin (M3), 6-glc-7-gluco-coumarin (M4), 6-O-methyl-7-gluco-coumarin (M5) and 6-O-methyl-7- sulf-coumarin (M6). Of which, M2 and M6 were novel metabolites. CONCLUSION:Aesculin can be transferred into aesculetin by human gut bacteria and is furth er modifiedby the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.