Abstract: CAT3 is a promising anti-brain tumor agent that has significant anti-tumor activity on Daoy or U87MG orthotopic xenograft in nude mice. This study was carried out to investigate the metabolic profiles of CAT3 in mouse/dog/human blood and microsome as well as in humanized recombinant enzymes. All animal care and experimental procedures were reviewed and approved by the Animal Ethics Committee of Chinese Academy of Medical Sciences. Our findings showed that CAT3 could be hydrolyzed to active metabolite PF403 by carboxylesterase, butyrylcholinesterase and serine hydrolase in mouse/dog/human blood. PF403 could be further metabolized to M1 oxidative dehydration product, M2 double oxidation dehydration product, M3 methylation oxidative dehydration product, M4 oxidation product and M5 demethylation product, which were mainly catalyzed by CYP1A2, 1A1, 2C9 and 3A4, and slightly by CYP2B6, 2C8, 2C19 and 2D6. Besides oxidative metabolism, PF403 also was transformed into glucuronylation metabolites GLU-PF403 by Phase Ⅱ enzymes UGT1A1, 1A3 and 1A9. Taken together, the metabolism of CAT3 was a multiple enzyme catalytic reaction. These results could provide valuable information for potential enzyme-mediated DDI in clinic studies.