Imigliptin has been reported as a novel dipeptidyl-peptidase-IV (DPP-4) inhibitor to treat type 2 Diabetes Mellitus (T2DM), and is currently being tested in clinical trials. In the first human clinical study, imigliptin was well tolerated and proved to be a potent DPP-4 inhibitor. Considering its potential therapeutic benefits and promising future, it is of great importance to study the metabolite profiles in the early stage of drug development. In the present study, a robust and reliable analytical method based on the ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method combined with MassLynx software was established to investigate the characterization of metabolites of imigliptin in human and rat plasma, urine and feces after oral administration. As a result, a total of 9 metabolites were identified in humans, including 6, 9 and 8 metabolites in human plasma, urine, and feces, respectively. A total of 11 metabolites were identified in rats, including 7, 10 and 8 metabolites in rat plasma, urine, and feces, respectively. In addition, 6 of the metabolites detected in humans and rats were phase I metabolites, including demethylation, carboxylation, hydroxylation and dehydrogenation metabolites, and 5 of the metabolites were phase II metabolites, including acetylation and glucuronidation. There was no human metabolite detected compared to those in rats. The major metabolites detected in human plasma (M1 and M2) were products resulting from acetylation, and hydroxylation followed by dehydrogenation. M1 was the major metabolite in rat plasma. M2 and the parent drug were the major drug-related substances in human urine. The parent drug was the major drug-related substances in rat urine. M2, M5 (hydroxylation product) and M6 (2×hydroxylation and acetylation product) were the predominant metabolites in human feces. M2 and M5 were the major metabolites in rat feces. In addition, renal clearance was the major route of excretion for imigliptin.