Abstract: Defensin, a small cationic peptide, is an antimicrobial peptide, exhibit broad-spectrum antibacterial activity. They spread widely in plants, invertebrate and vertebrate animals, which can rapidly kill bacteria, fungi and viruses etc. Bacteria and viruses limited the production of grouper (Epinephelus coioides). In this study, we cloned grouper -defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper -defensin cDNA was amplified by polymerase chain reaction (PCR) from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper. The 129 bp DNA fragment encoding mature grouper -defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPCIZA and transfected into Pichia pastoris GS115 expression host by electroporation. The genome of Pichia pastoris clone was extracted as template for screened the positive clone by using PCR. After grouper -defensin was secreted by GS 115 by small-scale culture, the recombinant grouper -defensin was induced by methanol for large scale. The supernatant was collected in 24h, 48h and 72h post induction for the recombinant protein detection. Subsequently, the grouper -defensin peptide was detected in the supernatant of transfected yeast by Western Blot analysis. The purified recombinant grouper -defensin crude extract from transfected P. pastoris showed antimicrobial activities against Escherichia coli and Aeromonas hydrophila. However, recombinant protein did not inhibit the growth of Gram-positive bacteria, such as Staphylococcus aureus and Micrococcus luteus. Bioactivity analysis indicated that the antibacterial activity of the recombinant grouper -defensin expressed in P. pastoris specific to Gram-negative bacteria. Grouper -defensin have antibacterial activity-specific to Gram-negative bacteria, this specificity is in correspondence with the environment of grouper.