Animal pharmacokinetic studies of sipoglitazar, a novel antidiabetic agent, showed that the deethylated metabolite (M-I) and the glucuronide conjugate of sipoglitazar (sipoglitazar-G) appeared to be the key metabolites in the elimination process. M-I was also measured as the main metabolite in the plasma of humans administered sipoglitazar. In vitro metabolic studies were performed to investigate the metabolic pathways from sipoglitazar to M-I in humans. The metabolic profile with human hepatocytes and hepatic microsomes indicated that M-I was not formed directly from sipoglitazar and that sipoglitazar-G was involved in the metabolism from sipoglitazar to M-I. Further studies of the metabolism of sipoglitazar-G revealed that the properties of the glucuronide conjugate and its metabolism are as follows: high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, and NMR analyses showed that sipoglitazar-G was composed of two glucuronides, sipoglitazar-G1, a β-1-O-acyl glucuronide, and sipoglitazar-G2, an α-2-O-acyl glucuronide. The stability study of these glucuronides suggested that sipoglitazar-G1 could be converted to sipoglitazar-G2 and sipoglitazar, but sipoglitazar-G2 could not be converted to sipoglitazar-G1. The oxidative metabolic study of sipoglitazar-G1 and -G2 with human hepatic microsomes and cytochrome P450-expressing microsomes revealed that M-I was formed only from sipoglitazar-G1, not from sipoglitazar-G2, and that CYP2C8 was mainly involved in this process. From these results, it is shown that the metabolic pathway from sipoglitazar to M-I is an unusual one, in which sipoglitazar is initially metabolized to sipoglitazar-G1 by UDP-glucuronosyltransferase and then sipoglitazar-G1 is metabolized to M-I by O-dealkylation by CYP2C8 and deconjugation. Sipoglitazar-G2 is sequentially formed by the migration of the β-site of sipoglitazar-G1.