Mouse beta defensin-1 (mBD-1) is a cationic 37-amino acid antimicrobial peptide with three conserved cysterine disulfied bonds. It exhibits a broad antimicrobial spectrum, but mBD-1 against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) is poorly understood. This study describes the mBD-1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released mature mBD-1. By constructing the expression plasmid (pET32a-mBD1), high yields of soluble mBD-1 fusion protein (0.67 g/L) could be obtained in E. coli and cleaved by enterokinase. The digested product was further purified and desalted with the final amount of pure mature mBD-1 being 0.14 g/L. Classical fungi growth inhibition assay showed clear antifungal activity against C. albicans and C. neoformans with IC50 of 5 and 2 mu M, respectively. The results show that the mBD-1 control fungal colonization through hyphal induction, direct fungicidal activity, and the activity is suppressed by increasing NaCl concentration. Successful expression of the mBD-1 peptide in E. coli offers a basis for further studying its antifungal mechanisms and may provide significance in developing this peptide to an antifungal drug.