Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the a-factor signal peptide, trypsin production (amidase activity) was improved to 177.85 +/- 2.83 U.mL(-1) in a 3-L fermenter (a 3.75-fold increase). Subsequently, all of the residues involved in autolysis that were identified by mass spectrometry were mutated and the resulting proteins were evaluated. In particular, the variant tbcf (K101A) demonstrated high stability and production (227.65 +/- 6.51 U.mL(-1) and 185.71 +/- 5.68 mg.L-1, respectively). The recombinant strain constructed here has great potential for large-scale production of active trypsin.