Abstract: To investigate metabolic conditions for bacteriocin production from Lactobacillus and its biological characteristics. The antimicrobial activity of the cell- free supernatant ( CFS) of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum was investigated by the agar diffusion assay.The results of orthogonal tests showed: when Lactobacillus casei was cultured for 42 h at 30℃ in the conditions of liquid volume of 20% and inoculum dose of 2%, the antimicrobial activity of the CFS came to the highest. The CFS of Lactobacillus casei culture was heat- stable, could maintain good antimicrobial activity at low p H conditions and was found to be sensitive to proteolytic enzymes such as trypsin, neutral protease and papain, but not pepsin. The action mode of the CFS was confirmed as bactericidal.The CFS showed a broad spectrum of antimicrobial activity against various species of gram- negative bacteria and some gram- positive bacteria, and also had a strong resistance to a variety of food- borne pathogens.These data showed, it could be developed as a feed additive for livestock and poultry breeding or natural food preservative for the food industry.