Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayer. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, cluadin-1, claudin-4 and ZO-1 from the intercellular junctions, and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4 or LBP by specific siRNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, an MLCK inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1were found to be highly phosphorylated on threonine residue in intact cell monolayer. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residue was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src, TLR4, LBP and MLCK-dependent mechanism.