Tissue factor pathway inhibitors (TFPIs) are Kunitz-type serine protease inhibitors that reversibly regulate the blood coagulation induced by tissue factor. TFPI family contain two members, TFPI-1 and TFPI-2. Recent studies have shown TFPI-1 and TFPI-2 also play important roles in innate immunity, however, the potential function of teleost TFPI are very limited. In this study, we characterized two TFPI (CsTFPI-1 and CsTFPI-2) molecules from half-smooth tongue sole (Cynoglossus semilaevis), examined their tissue distributions and expression patterns under pathogens stimulation as well as investigated the antibacterial activity of the C-terminal peptides. Quantitative real time RT-PCR analysis showed that constitutive CsTFPI-1 expression occurred, in increasing order, in head kidney, intestine, brain, spleen, liver, skin, gills, heart, and muscle; CsTFPI-2 was expressed, in increasing order, in the gills, intestine, skin, head kidney, liver, brain, spleen, muscle, and heart. Under Vibrio anguillarum, Streptococcus agalactiae and fish megalocytivirus stimulation, both CsTFPI-1 and CsTFPI-2 expression increased significantly in a manner that depended on the pathogen, tissue type, and infection stage, which suggested CsTFPI-1 and CsTFPI-2 play important roles in anti-bacterial and anti-viral infection. Finally, C-terminal peptides of CsTFPI-1 and CsTFPI-2, were synthesized and proved to have antibacterial effect against Micrococcus luteus that were independent of host serum. Take together, these results indicate that CsTFPI-1 and CsTFPI-2 play important roles in antimicrobial immunity of this fish.