Abstract: The objective of the present study was to clone, express, characterize the antimicrobial activity of duck avian βdefensin (AvBD) 4, and determine its mRNA distribution in tissues of duck. Reverse transcription polymerase chain reaction (RTPCR) was used to amplify duck AvBD4 gene from bursa of Fabricius of duck. Sequence analysis showed that the cDNA of duck AvBD4 consisted of 171 bp nucleotide, encoding a polypeptide of 56 amino acids. Homology analysis showed that duck AvBD4 shared high percentage of amino acid homology (73.2 %) with chicken AvBD4. The cDNA of duck AvBD4 mRNA was subcloned into EcoRⅠ and SalⅠ sites of pGEX6p1 vector to construct recombinant plasmid pGEXduck AvBD4.The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. It was demonstrated by SDSPAGE that a 32 kD protein which was equal to duck AvBD4 protein in molecular weight was highly expressed. The purified recombinant duck AvBD4 exhibited extensive antimicrobial activity against 11 strains of bacteria, including Grampositive and Gramnegative investigated. In high salt ions conditions, recombinant duck AvBD4 protein remained antibacterial activity against Staphylococcus aureus and Pasteurella multocida. In addition, the recombinant protein of hemolytic activity was extremely low. Additionally, real time PCR was used to determine tissue distribution of the gene. It was showed that AvBD4 mRNA was expressed limitedly in tissues of ducks. It was moderately expressed in the cecal tonsil, spleen, and bursa of Fabricius of ducks.