Bovine lactoferrin peptides are short peptides with broad spectrum antimicrobial activity produced by hydrolysis of bovine lactoferrin by pepsin. As a food-grade microorganism, Lactococcus lactis has a natural probiotic effect, and it is an ideal bacterial vector for expressing bovine lactoferrin peptide. [Objective] To investigate the antibacterial activity of bovine lactoferrin peptide expressed by the recombinant L. lactis pAMJ399-LFcinBA/MG1363. [Methods] We used the standard of bovine lactoferrin peptide to determine a quantitative standard curve to determine the concentration of the bovine lactoferrin peptide expressed in the supernatants of the recombinant strain, and used the Oxford Cup method to determine the inhibitory effects of the recombinant bovine lactoferrin peptide on 35 strains of bacteria including Escherichia coli and Staphylococcus aureus. And we used the broth microdilution method to determine the minimal inhibitory concentrations. We explored the effects of the recombinant lactoferrin peptide on a number of pathogenic bacteria using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. We used gel retardation assay and adhesion test to investigate the effects of the recombinant lactoferrin peptide on bacterial DNA and adhesion. We used CCK-8 to detect its toxic effects on RAW 264.7 cells and to determine the hemolytic rate of red blood cells in mice. [Results] The results showed that the concentration of the bovine lactoferrin peptide expressed in the supernatants of the recombinant strain was 24.39 μg/mL and the recombinant lactoferrin peptide showed inhibition on 25 strains of pathogenic bacteria used in the experiment. Its antibacterial concentration range was 16-128 μg/mL, but had no obvious inhibitory effect on 9 kinds of lactic acid bacteria and 1 strain of Enterococcus faecalis. It destroyed the integrity of E. coli, Staph. aureus, Pasteurella multocida and Salmonella pullorum, targeting the bacterial membranes. It could bind to bacterial DNA and inhibit bacterial adhesion to Caco-2 and IPEC cells. The recombinant lactoferrin peptide had no obvious cytotoxic effect on mouse red blood cells and RAW 264.7 cells.[Conclusion] The recombinant bovine lactoferrin peptide had the same antibacterial activity as the standard of bovine lactoferrin peptide. It could inhibit or kill bacteria by targeting the membranes of bacteria, intracellular nucleic acids and inhibiting the adhesion of bacteria to normal cells, exerting its broad-spectrum antibacterial activity, and had no obvious cytotoxicity to eukaryotic cells.