Human tissue factor pathway inhibitor-2 (hTFPI-2) has three kunitz domains whose structure and function are unclear. We expressed the first kunitz domain of hTFPI-2 (hTFPI-2/KD1) as functional form using Pichia pastoris and investigated its characterization. In the experiment, hTFPI-2/KD1 can inhibit the plasmin and trypsin activity and the Ki of hTFPI-2/KD1 towards plasmin (30nM) and trypsin (50nM) was determined as 10 and 7nM by chromogenic assay, respectively. hTFPI-2/KD1 can also inhibit MMP-2 and MMP-9 in zymography assay. Furthermore, the inhibition of hTFPI-2/KD1 to the Matrigel invasion by HT-1080 is also described. This study provides a method to produce hTFPI-2/KD1 efficiently and some insights into the structure and function of hTFPI-2/KD1.