4-({4-[(2-hydroxy-ethyl)-methyl-carbamoyl]-phenyl}-quinolin-8-yl-methylene)-1-thiazol-4-ylmethyl-piperidinium (compound I) is a selective agonist of δ-opioid receptor developed for the treatment of depressive and anxiety disorders. The in vitro biotransformation studies using rat, dog, and human hepatocytes showed that the metabolites detected in human hepatocytes were also found in either rat or dog hepatocytes. M1 (N-dealkylation), M2 (N-demethylation), and M4 (carboxylic acid metabolite) were major phase I metabolites observed in all three species. Human CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the formation of M1 and M2 in human liver microsomes. After single oral administration of [¹⁴C]compound I, the major elimination route for [(¹⁴C]compound I and its metabolites in rat was through feces with 92.9% recovery. The results from the bile duct-cannulated study revealed that a minimum of 51% of administered dose was absorbed in rats. The pharmacokinetic analysis using unlabeled parent drug showed that compound I was rapidly absorbed and exhibited a mean apparent terminal half-life of approximately 2.7 h. A total of 15 metabolites of compound I were detected and profiled in rat urine, bile, and feces. In rat bile, compound I accounted for <1.5% of the excreted dose, suggesting that compound I underwent extensive metabolism before elimination. The structures of metabolites were elucidated by high-resolution tandem mass spectrometry. M1, M4, and M6 were the most abundant metabolites observed in rat bile. Only a low level of parent [¹⁴C]compound I was observed in rat plasma.