The in vivo and in vitro metabolism of mangiferin was systematically investigated. Urine, plasma, feces, contents of intestinal tract and various organs were collected after oral administration of mangiferin to healthy rats at a dose of 200mg/kg body weight. For comparison, mangiferin was also incubated in vitro with intestinal flora of rats. With the aid of a specific and sensitive liquid chromatography coupled with electrospray ionization tandem hybrid ion trap mass spectrometry (LC-ESI-IT-MS(n)), a total of thirty-three metabolites of mangiferin were detected and their structures were tentatively elucidated on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The biotransformation pathways of mangiferin involved deglycosylation, dehydroxylation, methylation, glycosylation, glucuronidation and sulfation.