P-glycoprotein is an efflux pump belonging to the ATP-binding cassette super-family that influences the bioavailability and disposition of many drugs. Mammary epithelial cells express various drug transporters including P-glycoprotein, albeit at low level during lactation. During inflammatory reactions, which can be associated with changes in epithelial barrier functions, pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) are elevated in milk and serum. In this study, the role of TNF-α in the regulation of P-glycoprotein was determined in cultured BME-UV cells, an immortalized bovine mammary epithelial cell line. The protein production of P-glycoprotein and mRNA expression of bABCB1, the gene encoding P-glycoprotein, were increased after 24 hours of TNF-α exposure. The highest observed effects for TNF-α on the regulation of P-glycoprotein was after 72 hours of exposure. Protein and mRNA expression also significantly increased after 120 hours of TNF-α exposure, but it was lower than the level that observed in the cells exposed to TNF-α for 72 hours. The apical to basolateral flux of digoxin, a P-glycoprotein substrate, was decreased in the TNF-α-exposed epithelium. This effect was reversed when verapamil or ketoconazole, compounds known to interact with P-glycoprotein, were added together with digoxin into the donor compartment. Probenecid, a compound known to interact with organic anion transporters, but not P-glycoprotein, did not increase the flux of digoxin. This model has important implications for understanding the barrier function of the mammary epithelium and provides insight into the role of P-glycoprotein in the accumulation and/or removal of xenobiotics from milk and/or plasma.