Insulin-like peptide 6 (INSL6) is a newly identified peptide hormone belonging to the insulin/relaxin superfamily. It is predominantly expressed in the testes and responsible for spermatogenesis. It also shows potential for the treatment of benign prostate hyperplasia. For further basic and preclinical studies, significant quantities of INSL6 peptide are needed. In the present work, we designed and recombinantly expressed a single-chain human INSL6 precursor in Escherichia coli. After purification, in vitro refolding, and cyanogen bromide cleavage, the single-chain precursor was converted to mature two-chain INSL6 peptide. So far, the receptor of INSL6 is remained unknown. To identify its receptor, site-specifically biotin-labeled INSL6 analogues are useful. Thus, we designed and recombinantly prepared a two-chain INSL6 analogue carrying an Avi-tag at the N-terminal of B-chain. Subsequently, biotin moiety was covalently attached to the Avi-tag of the analogue catalyzed by biotin ligase. Circular dichroism spectroscopy suggested that the recombinant INSL6s adopted insulin-like fold. Our present work provided an efficient approach for the preparation of the recombinant INSL6 peptide and its analogues for further studies of this important peptide. (C) 2011 Elsevier Ltd. All rights reserved.