To study the safety of Aconitum medicinal herbs in clinic and identify Aconitum alkaloids poisoning in forensic medicine, Aconitum alkaloids and their metabolites were separated and identified in human urine by liquid chromatography-electrospray ionization-multi-stage mass spectrometry (LC-ESI-MS(n)) and chemical pathway of metabolism was investigated. The alkaloids and their metabolites in the urine sample were extracted with solid-phase cartridges and separated by HPLC with acetonitrile-water-formic acid (40:60:0.5) mobile phase. Structures of five metabolites and three parent Aconitum alkaloids were identified with multi-stage mass spectrometry data through comparison with authentic substances as aconitine (M(1)), mesaconitine (M(2)), hypaconitine (M(3)), benzoylaconine (M(4)), benzoylmesaconine (M(5)), benzoylhypaconine (M(6)), 16-O-demethylaconitine (M(7)) and 16-O-demethylhypaconitine (M(8)), respectively. Among them, M(8) was identified and reported for the first time. Metabolic pathways of Aconitum alkaloids in human body were proposed.