Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation system. Recent studies showed that TFPI could also inhibit the proliferation of vascular smooth muscle cells. Studies has suggested that TFPI may find wide applications for the therapy of various diseases such as sepsis, DIC, vascular stenosis, atherosclersis, and miocardial infarction, etc. However, the rapid clearance of TFPI from the circulation remains to be one of major drawbacks to its clinical application. The aim of the present study is to prolong the half-life of recombinant TFPI while maintaining its biological activities by reducing the interaction between TFPI and cell surface, which is mediated by the negative charges on the membrane of the cells and the positive charge on the molecule of TFPI. For this purpose, three rTFPI mutants, m₀TFPI, m₁TFPI, and m₂TFPI, were generated by introducing mutations in the DNA sequences coding for the C-terminal amino acid sequences. The clearance rate of the experimental group (three rTFPI mutants) and the control group (wild-type TFPI, namely mTFPI) in rats was investigated. The inhibitory function on FXa and the heparion binding capacity of the TFPI mutants were also studied. It was shown that at 10 min after intravenous injection, (59.06 ± 13.54) % of the injected mTFPI remained in the circulation, while (71.14 ± 13.04) % of the m₀TFPI, (77.99 ± 2.53) % of the m₁TFPI, and (82.95 ±11.36) % of the m₂TFPI were still in the circulation. The relative half-life of m₀TFPI, m₁TFPI and m₂TFPI were prolonged 0.5, 0.9, and more than 1 times respectively in comparison with that of mTFPI, while their FXa inhibiting activity and heparin-binding capacity were little changed.