The blood-milk barrier of the bovine mammary gland prevents an intermixture of blood and milk. During mastitis, the permeability of the blood milk barrier is increased, which is reflected by an exchange of blood constituents into milk, and vice versa. The aim of this study was to investigate the role of mammary epithelial cells in the regulation of blood-milk barrier alteration during mastitis induced by cell wall components of Escherichia coli (LPS) and Staphylococcus aureus (LTA). Low passage primary bovine mammary epithelial cells (bMEC) from 3 different cows were grown separately on Transwell inserts. The level of integrity of the epithelial barrier was measured by means of transepithelial electrical resistance (TEER), as well as by diffusion of the fluorophore Lucifer Yellow (LY) across the cell layer. The formation of tight junctions between adjacent epithelial cells was examined by immunofluorescence staining of zona occludens-1 and by transmission electron microscopy. The cultured cells formed tight cell layers sealed by tight junctions. The barrier integrity was reduced after 3 h (P < 0.05; paired t-test relative to unchallenged cells) in response to 500 µg/mL LPS from E. coli, and after 7 h in response to 20 mg/mL LTA from S. aureus. At these dosages, the fluorescence values of LY in the apical compartment of the Transwell insert dropped within 24 h from 5386 to 560 ± 235 RFU in response to LPS, and from 5386 to 1416 ± 144 RFU in response to LTA. No significant changes in barrier permeability were observed in response to 200 μg/mL LPS, or to 2 mg/mL LTA Although LPS and LTA affected the barrier permeability most likely due to an opening of the tight junctions, LPS additionally caused considerable cell damage reflected by increased LDH concentrations in cell culture medium (P < 0.05). These results confirm a pathogen-specific impairment of the blood-milk barrier during mastitis, which involves differences in cell degradation.