Rattusin is an alpha-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations. Therefore, to develop new antibiotics based on rattusin, structural and functional studies of rattusin should be performed. For this purpose, large amounts of linear rattusin precursor must be obtained through appropriate preparation methods. Therefore, we established a mass production technique for linear rattusin by using recombinant protein expression and purification procedures. We verified that structure and activity of the recombinant rattusin are identical to the chemically synthesized rattusin. The described method for producing recombinant rattusin provides a high yield of rattusin, which can be used to study the biochemical and functional properties of rattusin and for the development of rattusin-based peptide antibiotics.