Metabolism of melatonin (MEL) in mouse was evaluated through a metabolomic analysis of urine samples from control and MEL-treated mice. Besides identifying seven known MEL metabolites (6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin), principal components analysis of urinary metabolomes also uncovered seven new MEL metabolites, including MEL glucuronide, cyclic MEL, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin; 5-hydroxyindole-3-acetaldehyde, di-hydroxymelatonin and its glucuronide conjugate. However, N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine and N(1)-acetyl-5-methoxy-kynuramine, known as MEL antioxidant products, were not detected in mouse urine. Metabolite profiling of MEL further indicated that 6-hydroxymelatonin glucuronide was the most abundant MEL metabolite in mouse urine, which comprised 75, 65, and 88% of the total MEL metabolites in CBA, C57/BL6, and 129Sv mice, respectively. Chemical identity of 6-hydroxymelatonin glucuronide was confirmed by deconjugation reactions using beta-glucuronidase and sulfatase. Compared with wild-type and CYP1A2-humanized mice, Cyp1a2-null mice yielded much less 6-hydroxymelatonin glucuronide (approximately 10%) but more N-acetylserotonin glucuronide (approximately 195%) and MEL glucuronide (approximately 220%) in urine. In summary, MEL metabolism in mouse was recharacterized by using a metabolomic approach, and the MEL metabolic map was extended to include seven known and seven novel pathways. This study also confirmed that 6-hydroxymelatonin glucuronide was the major MEL metabolite in the mouse, and suggested that there was no interspecies difference between humans and mice with regard to CYP1A2-mediated metabolism of MEL, but a significant difference in phase II conjugation, yielding 6-hydroxymelatonin glucuronide in the mouse and 6-hydroxymelatonin sulfate in humans.