Abstract: Matrix-assisted laser desorption/ionization mass spectrometry was used to direct analyze the metabolites of aconitine under rat intestinal bacteria. Only simple sample preparation was needed prior to the MS experiment. CHCA given the good signals was screened as the matrix for the analysis of metabolites. Aconitine can be converted into its metabolites by rat intestinal bacteria such as monoester-diterpenoid alkaloids, diester-diterpenoid alkaloids and lipo-alkaloids by deacetylation, dehydroxylation, demethylation and esterification.