Objective To determine the effect of vitamin K metabolize associated gene CYP4F2 on the activation of vitamin K dependent coagulation factor Ⅸ (FⅨ). Methods The pIRES plasmid containing CYP4F2-cDNA fragment and FⅨ-cDNA fragment was constructed, and enzyme digestion was used to verify the products. The plasmids pIRES-FⅨ-CYP4F2, pIRES-FⅨ and pIRES(control plasmid) were transfected into LO2 cells respectively with Lipofectamine 2000 transfection reagent. Western blotting was used to detect the expression of CYP4F2 and FⅨ.The activity of FⅨa was detected by FⅨa activity assay kit. Results The results of enzyme digestion indicated that the pIRES-FⅨ-CYP4F2 plasmid was successfully constructed. The plasmids pIRES-FⅨ and pIRES-FⅨ-CYP4F2 were successfully transfected into LO2 cells. FⅨa activity was significantly higher in the supernatants and cell lysates from the FⅨ-overexpressed LO2 cells than the control cells, but the activity was significantly reduced in the LO2 cells with the over-expression of FⅨ and CYP4F2 than the cells with the over-expression of FⅨ. Conclusion The over-expression of CYP4F2 exerts inhibitory effect on the activation of vitamin K dependent FⅨ.