Platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid mediator in a variety of physiological events. PAF is also involved in various pathological events including allergy and inflammation. PAF-acetylhydrolase (PAF-AH) hydrolyzes PAF to produce inactive lyso-PAF. Thus, overproduction of PAF-AF will be useful for the therapeutic valuation of the enzyme. In this study, we established an overproduction method of bovine PAF-AH in Escherichia coli system. We used bovine mammary gland for cDNA cloning. The cDNA had two mismatches of amino acid sequences (Thr-247 to Met and Ile-431 to Thr) compared with the previously reported PAF-AH cDNA (bovine spleen, NM_174578). The recombinant PAF-AH of 43 kDa in molecular size reacted with human PAF-AH polyclonal antibody and showed a strong PAF-AH enzyme activity in an in vitro assay system. The recombinant PAF-AH produced by this study can be applied for various experiments including in vivo models to test its protective activity against PAF-related diseases.