Mammalian beta-defensins are an important family of host defense peptides with diverse functions. Surprisingly most of the mammalian beta-defensin genes are revealed preferentially expressed in the male organs. There is a pressing need to understand how the ample defensin repertoires work in both host defense and fertility with an aim to overcome antibiotic resistance of pathogens and reproductive problems. The biggest obstacle is the production of beta-defensin peptides as beta-defensins are small, antimicrobial and multi-disulfide molecules. In this study, the well documented HBD2, function-unknown RBD1 and function-partly-known rBin1b are successfully expressed and assayed. This approach overcomes the difficulties in beta-defensin production and provides a convenient and economical peptide-production platform to elucidate the antimicrobial activities and clinical prospects of beta-defensins. In the strategy of recombinant expression, this approach may be the best to develop the "natural" peptide pools for both host defense and fertility in a cost-effective manner.