β-lapachone (β-Lap) is an NAD(P)H: quinone oxidoreductase 1 (NQO1) target anti-tumor drug candidate in phase Ⅱ clinical trials.The present study aimed to uncover the metabolic characteristics, enzyme kinetics, and the enzyme isoforms of β-Lap in the human liver and intestine in vitro.NQO1-mediated quinone reduction and subsequent glucuronidation is the predominant metabolic pathway for β-Lap in human; a pair of regioisomers (M1 and M2) of reduced β-Lap glucuronides were the major metabolites found from human S9 incubations.The overall glucuronidation clearance of β-Lap in human liver S9 was 4754.90μL/min/mg protein about 8.1 fold of that in the human intestinal S9.Recombinant UDP-glueuronosyitransferases (UGTs) screening, correlation analysis, enzyme kinetics, and chemical inhibition study were performed to determine the UGT isoforms involved in β-Lap metabolism.UGT1A7,UGT1A8, and UGT1A9 were predominantly responsible for the formation of M2 and UGT2B7 was the main isoform contributing to the formation ofMl, suggesting a regioselective glucuronidation of reduced quinone by UGTs.It was of great interest to find that β-Lap can undergo non-enzymatic catalysis of two-electron reduction, which may at least partially explain the poor selectivity of β-Lap at high concentration and long-time exposure towards NQO1 catalyzed quinone reduction.In conclusion, this represents the first study to the enzymes involved in the metabolism of β-Lap, which is not only important for the understanding of its metabolism, but the anti-tumor efficacy as well.