OBJECTIVE To inv estigate the in vitro inhibitory effects of acteoside on cytochrome P 450（CYP）enzymes in liver microsomes of rats. METHODS Using probe substrates method ，acteoside（0.1，0.3，1，3，10，30 μmol/L）was incubated with probe substrates phenacetin ，mephentoin，diclofenac，coumarin，dextromethorphan and testosterone （substrates of CYP 1A2， CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）in liver microsomes of rats. Another blank control group and positive inhibitor group [ α-naphthoflavone，ticlopidine，sulfabendazole，pilocarpine，quinidine and ketoconazole （inhibitors of CYP 1A2，CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）] were set up. Using indapamide as the internal standard ， the contents of corresponding metabolites （acetaminophen， 4-hydroxymephenytoin， 4-hydroxydiclofenac，7-hydroxycoumarin，dextran，6 β-hydroxytestosterone） were detected by ultra high performance liquid chromatography-tandem mass spectrometry . The IC 50 values were calculated by GraphPad v 8.0 software. By computer molecular docking technology ，acteoside and positive inhibitors were molecularly docked with the CYP enzyme ，and the binding mode and strength of the two molecules were analyzed. RESULTS The IC 50 values of acteoside to CYP 1A2 and CYP 2A6 enzymes were more than 30 μmol/L，and those of acteoside to CYP 2D6，CYP2C19，CYP2C9 and CYP 3A4 enzymes were 24.87，21.52，12.56 and 7.55 μmol/L，respectively. The hydrogen bond and hydrophobic force could form between acteoside and CYP 3A4 enzyme，and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP 3A4 enzyme. The binding free energy of acteoside and ketoconazole to CYP 3A4 enzyme were － 10.2 and － 12.4 kcal/mol （1 kcal/mol＝4.19 kJ），respectively. CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP 3A4 enzyme in liver microsomes of rats ，and its affinity is equivalent to that of positive inhibitor ；the compound shows weak inhibitory effect on other 5 CYP enzymes.