The difficulty in the purification of bioactive peptide limited its application in food, drug and cosmetic industry. Here we report a new strategy for the recovery of two peptides employing glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL), which shows strong specificity for Glu residue. Human glucagon and human beta-defensin-2 (HBD-2) were peptides without Glu residue, and Glu residue was introduced between affinity tag and target peptide as recognition site of GSE-BL. Tagless human glucagon with the same HPLC retention time as native human glucagon and mature HBD-2 with antibacterial activity and cytotoxicity were obtained after GSE-BL treatment. This strategy has great potential in the recovery of bioactive peptide without Glu residue, thus facilitating large scale preparation of peptide and widening the application of bioactive peptide. (C) 2013 Elsevier Inc. All rights reserved.