Background: The herbal-pair, Kansui and Licorice, belongs to the "eighteen incompatible medicaments" category of traditional Chinese medicine. Kansuiphorin C (KC) is the main toxic component of Kansui. The main component of licorice is glycyrrhizic acid, which is hydrolyzed to glycyrrhetinic acid. Currently, the synergistic mechanism between Kansui and Licorice is unclear. Methods: Rat liver microsomes were used in this experiment, HPLC was used to detect the contents of KC, glycyrrhizic acid, and glycyrrhetinic acid to determine whether these compounds are metabolic substrates of CYP450. A control group with isozyme inhibitors was also employed to reveal the isozyme subtypes involved in compound metabolism. To further explain the induction or inhibitory effect of the above compounds on liver microsomal enzymes, enzyme activity was indirectly revealed based on changes in the contents of known metabolites of CYP2E1, CYP2C9, and CYP3A4. Results: KC and glycyrrhetinic acid were metabolic substrates of CYP450. CYP2E1 and CYP2C9 are mainly involved in the partial metabolism of glycyrrhizic acid in the liver. CYP2E1 and CYP3A4 are mainly involved in the partial metabolism of glycyrrhetinic acid in the liver. CYP2E1, CYP2C9, and CYP3A4 did not play a major role in the metabolism of KC. KC had little effect on the metabolism of glycyrrhizic acid and glycyrrhetinic acid. Glycyrrhizic acid, glycyrrhetinic acid, and KC induced CYP3A4 and inhibit CYP2E1. Both glycyrrhizic acid and glycyrrhetinic acid could inhibit the induction of CYP3A4 after combination with KC. KC with glycyrrhizic acid could synergistically inhibit the activity of CYP2E1, while KC with glycyrrhetinic acid could synergistically induce the activity of CYP2E1 Conclusion: KC and glycyrrhetinic acid were metabolic substrates of CYP450. KC, glycyrrhizic acid and glycyrrhetinic acid have different inducing and inhibiting effects on CYP450 enzyme.