Bromopride (BRP) has been utilized clinically for treatment of nausea, vomiting and gastro-intestinal motility disorders. The pharmacokinetics of BRP have been characterized in dogs and humans; however, the metabolic profile of BRP has not been well studied. The present study was aimed at better understanding BRP metabolism across species. We investigated biotransformation of BRP in mouse, rat, rabbit, dog, monkey, and human hepatocytes with the help of LC-MS(n) and accurate mass measurement. Mice, rats, dogs, and monkeys are relevant in drug discovery and development as pre-clinical species to be compared with humans, whereas rabbits were efficacy models for BRP. Overall, twenty metabolites of BRP were identified across hepatocytes from the six species. Monkeys offered the most coverage for humans, in terms of number of metabolites identified. Interestingly, M14, an N-sulfate metabolite of BRP, was identified as a human-specific metabolite. BRP metabolism had only been reported in dog plasma and urine, historically. Our investigation is the first documentation of in vitro metabolism of BRP in the six species reported here. Metabolites M1, M2, M4-M10, M12, M13, and M15-M20 have not been previously reported. In summary, this report documents seventeen metabolites of BRP for the first time, thus providing a deeper insight into the biotransformation of BRP.