An inactivated mutant of matriptase serine protease domain (S195A) was constructed, and the recombinant protein in Pichia pastoris was expressed. After captured by anion exchange chromatography, the recombinant protein was further purified by gel-filtration chromatogram column and resource Q anion exchange column with high purity. High quality crystals of this inactivated protein were obtained by sitting-drop vapor diffusion. The results showed that the single point mutant increased the level of matriptase compared with the wild type. This inactivated mutant forms stable complex with its inhibitor HAI-1, although it does not possess catalytic activity. Crystals of this inactivated protein were diffracted to 1.48 ?. The mutant has the same conformation as the wild type.