Rationale: Sanguinarine (SAN), as the main active component of a traditional Chinese veterinary medicine, have been widely used in animal husbandry and breeding industry. However, the metabolites of SA are still uncertain. Therefore, This research aimed to investigate the metabolites of SA based on rats in vivo. Methods: Blood, feces and urine of rats were collected after oral administration of 40 mg/kg SAN. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was employed to identify the metabolites of SAN. The elemental composition of sanguinarine metabolites was inferred by analyzing their exact molecular weight, and the metabolites’ structure were predicted based on their fragment ions and cleavage pathways. Results: A total of 12 metabolites were identified, including three metabolites in plasma, four in urine and nine in feces. Accord to the possible metabolic pathways deduced in this study, SAN was mainly metabolized by reduction, oxidation, demethylation, hydroxylation and glucuronidation. Conclusions: The present research have summarized the metabolism of SAN in rats, which is helpful to further study on metabolic mechanism of SAN in vivo and in vitro.