Vabicaserin is a potent 5-hydroxytryptamine(2C) agonist that is currently being developed for the treatment of the psychotic symptoms of schizophrenia. In this study, in vitro and in vivo metabolism of vabicaserin was evaluated in mice, rats, dogs, monkeys, and humans, and the structures of the metabolites were characterized by liquid chromatography/mass spectrometry and NMR spectroscopy. Vabicaserin underwent three major metabolic pathways in vitro: NADPH-dependent hydroxylation, NADPH-independent imine formation, and carbamoyl glucuronidation. After a single oral dose, vabicaserin was extensively metabolized in animals and humans, and its metabolites were mainly excreted via the urine in mice and rats. Along with the metabolites observed in vitro, secondary metabolism via oxidation and conjugation of the primary metabolites generated from the above-mentioned three pathways yielded a number of additional metabolites in vivo. Carbamoyl glucuronidation was the major metabolic pathway in humans but a minor pathway in rats. Although carbamoyl glucuronidation was a major metabolic pathway in mice, dogs, and monkeys, oxidative metabolism was also extensive in these species. Hydroxylation occurred in all species, although different regional selectivity was apparent. The imine pathway also appeared to be common to several species, because vabicaserin imine was observed in humans and hydroxyl imine metabolites were observed in mice, rats, and dogs. A nitrone metabolite of vabicaserin was observed in dogs and humans but not in other species. In conclusion, the major metabolic pathways for vabicaserin in humans and nonclinical safety species include carbamoyl glucuronidation, hydroxylation, formation of an imine, and a nitrone.