Serine protease inhibitors are essential for host physiological and immunological activities in insects. Analyzing the amino-acid sequence of a cDNA coding for a serine protease inhibitor in Bombyx mori (BmSPI), we found that BmSPI contained three homologous domains with a conserved sequence of C-X(3)-C-X(9)-C-X(6)-Y-X(7)-C-X(3)-C-X(11)-C similar to that of Kazal-type serine protease inhibitors, suggesting BmSPI as a new member of the Kazal-type serine protease inhibitor family. To characterize the three-domain Kazal-type inhibitor from silkworm pupae, the recombinant protein was expressed in Escherichia coli BL21 (DE3) Star. After purification with affinity and reversed-phase chromatographies, the recombinant BmSPI with a molecular mass of 33.642 Da was shown to be a specific subtilisin A inhibitor. Further studies indicated that the K(i) value of the recombinant BmSPI was 3.35 nM and the inhibitor seemed to form a 1:1 complex with subtilisin A. This is a first description of the structure and characterization of Kazal-type inhibitor with three domains cloned from silkworm pupae, B. mori.