Objective To compare the effects of human Trypsin-1 signal peptide and pro-peptide on the expression and secretion efficiency of human Interleukin-25 from mammalian cells. Results The signal peptide and combined signal peptide-pro-peptide sequence of human Trypsin-1 improved the secretion of human IL-25 from 1.7 to 3.2 mu g/ml and 1.7 to 8.2 mu g/ml, respectively. Deletion analysis identified the minimal Trypsin-1 derived secretion domain that maintains improved human Interleukin-25 production and secretion. The presence of Trypsin-1 pro-peptide sequence does not affect the function of secreted human Interleukin-25. Conclusion The Trypsin-1 signal peptide-pro-peptide sequence increased human IL-25 expression and secretion in mammalian cells by fivefold.