Defensins are abundant in cells and tissues that are involved in host defense against microbial infections. zfDB3 (zebrafish β-defensin 3) is one of 3 copies of defensin β-like genes from zebrafish ( Danio rerio ). Here we focus on mzfDB3 , which is the gene encoding for the zebrafish β-defensin 3 mature peptide. A codon-optimized mzfDB3 gene with a 6×His-tag at the 3′-end was inserted into the pPICZαA expression vector and transformed into Pichia pastoris X-33 cells. The recombinant zebrafish β-defensin 3 mature peptide ( rmzfDB3 ) was induced with 1.0% methanol at 29°C for 72 h and purified by immobilized metal affinity chromatography. MALDI-TOF/TOF analysis confirmed the expected purified product ( rmzfDB3 , 5.9 kDa). Fermentation supernatant, which contained rmzfDB3, showed... antibacterial activity against Grampositive (i.e., Listeria monocytogenes, Staphylococcus aureus , and Bacillus cereus ) and Gram-negative (i.e., Escherchia coli BL21, Vibrio parahaemolyticus , Salmonella lignieres , and Pseudomonas aeruginosa ) bacteria.