Tissue factor (TF) plays an important role in the pathogenesis of atherosclerotic, sepsis and disseminated intravascular coagulation (DIC). TF pathway is therefore an attractive therapeutic target in a number of disease states. Here two TF mutants were developed and named MCsTF and MFsTF, in which the amino acids of active sites were mutated. Both of them were expressed in E.coli and used to inhibit TF pathway through competitive FVII/VIIa binding with TF. The results indicated that rMCsTF almost lost all activities of FX activation and procoagulation, and rMFsTF lost 90% activity. The specific catalytic constant ( k ( cat )/ K (m)) of FX activation by the complex formed by FVIIa with rMCsTF or rMFsTF were 2.0% and 3.7%, respectively, compared to that of rsTF. The inhibition effects of the mutants were studied in vitro, and it appeared that the prothrombin time were prolonged in a dose-dependent manner. Therefore, these mutants of TF may become new kind of specific inhibitors of TF pathway, as a promising drug for the treatment of patients with over-expression of TF.