Lipocalin-2 (LCN2) is a 25-kDa protein in the lipocalin family that shows antibacterial activity. The aim of this study was to clone human LCN2 into the mammalian-specific pIRES2-AcGFP1 vector and to determine the antibacterial activity of the recombinant pIRES2-AcGFP1-hLCN2 plasmid against major bacterial pathogens that cause bovine mastitis. The amplified hLCN2 was successfully cloned into the vector, and the plasmid was transfected into bovine mammary epithelial stem cells using nucleofection. Immunochemistry, reverse transcription polymerase chain reaction, and ELISA were used to monitor and quantify hLCN2 expression in the cells. After 1 day of incubation, the concentration of hLCN2 secreted by cells in the medium (6.85 ± 0.11 μg) was higher than that stored within the cells (22 ± 2.52 ng). After 4 days of culture, the amount of hLCN2 was 300-fold higher in the medium than within the cells. The recombinant clone showed potential antibacterial activity against mastitis-causing bacteria, with greater inhibitory activity against Escherichia coli than against Staphylococcus aureus. Antibacterial activity remained strong after 4-5 h of culture. Thus, pIRES2-AcGFP1 was identified as an efficient vector for the delivery of the target gene to the mammary cells, and recombinant hLCN2 showed strong antibacterial activity against major mastitis-causing bacteria.