Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori, and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/ MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human liver microsomes. When licoricidin was incubated at 0-25 µM with CYP probes for 60 min at 37 o C, it showed potent inhibitory effects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4'-hydroxylation with half maximal inhibitory concentration (IC 50 ) values of 3.4 and 4.0 µM, respectively. The inhibition mode of licoricidin was revealed as competitive, dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect of licoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with IC 50 values of 4.5 and 0.73 µM, respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activity could be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B-and CYP2C9-mediated metabolism.