Abstract: The study was conducted to clone bovine tracheal antimicrobial peptide gene (TAP) from bovine tracheas, and recombinant bovine TAP protein was expressed in E. coli. Its antimicrobial activity were determined. In addition, the tissue distribution of the TAP gene in bovine was investigated. The mRNA of bovine TAP was cloned from tracheas of Holstein cow by RT-PCR. Based on the analysis of the sequence of bTAP and the codon preference in E.coli expression system, the bovine TAP gene was synthesized and inserted into vector pET32a(+) to construct plasmid pET32a(+)-TAP. The plasmid was expressed in E. coli BL21(DE3) cell by induction of IPTG. The recombinant fusion protein was purified, and determined by SDS-PAGE and Western Blot. Antimicrobial activity of the recombinant fusion protein were measured by filter paper method. Tissues distribution of the TAP gene has been demonstrated in bovine by RT-PCR. The full length cDNA of bovine TAP was consisted of 114 bp, encoding 38 amino acid residues. Western Blot results showed that the recombinant proteins were expressed in E. coli with molecular weight of 24 kD, exists in soluble form and accounted for 52.1% of the whole bactera proteins. A single band appeared in Western Blot detection. The content of 0.115 μg·μL-1 of the purified TAP protein had antibacterial activity against bovine Sta.Aureus in vitro. Bovine TAP mRNA was widely expressed in the tissues of bovine, such as throat, lungs, trachea, small intestine, liver, heart, oral mucosa, and it was micro-expressed in the nasal mucosa, lingual mucosa and almost not expressed in the skin. The results showed that bovine TAP protein was highly expressed in E. coli, and can be used for transgenic dairy cow which anti-mastitis and developing the product of genetic engineering in future.