Objective To construct the recombinant retroviral vector capable of expressing human tissue factor pathway inhibitor and GFP in rat endothelial progenitor cells (EPCs). Methods Full length TFPI cDNA obtained from pIRES-TFPI by PCR amplification was digested with EcoRI and Xhol restriction enzymes and subsequently inserted into pMSCV-IRES-GFP expression vector to create the recombinant bicistronic retroviral vector pMSCV- TFPI- IRES -GFP encoding both TFPI and GFP . The recombinant plasmid was identified with restrictive endonuclease digestion and DNA sequencing. The recombinant plasmid was transfected into 293T and the supernatant containing packaged recombinant retroviral particles was collected and used to infect the EPCs isolated from rat bone marrow. TFPI mRNA was measured by RT-PCR and the amount of TFPI protein secreted from the transfected cells was determined by ELISA. GFP expression in the infected cells was analyzed by fluorescent microscopy and fluorescence activated cell sorting (FACS). Results Restriction endonuclease mapping and DNA sequencing confirmed the in-frame insertion of TFPI cDNA into the constructed vectorpMSCV-TFPI-IRES-GFP. Both RT-PCR and ELISA analysis demonstrated increased TFPI expression in the EPCs infected with pMSCV-TFPI-IRES-GFP. FACS analysis demonstrated that the transduction efficiency of EPCs with pMSCV-TFPI-IRES-GFP in vitro was over 90%. Conclusion pMSCV-TFPI-IRES-GFP could be effectively expressed in cultured EPCs and may provide a useful tool for further study on the application of TFPI in the prevention of restenosis.