While it is clear that bile acid accumulation is the major initiator of fibrosis caused by cholestatic liver disease, endotoxemia is a common side effect. However, the depletion of hepatic macrophages with gadolinium chloride blunts hepatic fibrosis. Since endotoxin is a key activator of hepatic macrophages, this study was designed to test the hypothesis that LPS signaling through CD14 contributes to hepatic fibrosis caused by experimental cholestasis. Wild-type mice and CD14 knockout mice (CD14-/-) underwent sham operation or bile duct ligation and were sacrificed three weeks later. Measures of liver injury, such as focal necrosis, biliary cell proliferation, inflammatory cell influx were not significantly different among the strains three weeks after bile duct ligation. Markers of liver fibrosis such as Sirius red staining, liver hydroxyproline and -smooth muscle actin expression were blunted in CD14-/- mice compared to wild type mice following bile duct ligation. Despite no difference in lymphocyte infiltration, macrophage/monocyte activation marker OX42 (CD11b) and oxidative stress/ lipid peroxidation marker 4-hydroxynonenal were significantly up-regulated in wildtype mice following bile duct ligation but not in CD14-/- mice. Increased profibrogenic cytyokine mRNA expression in the liver after bile duct ligation was significantly blunted in CD14-/- mice compared to wildtype. The hypothesis that LPS was involved in experimental cholestatic liver fibrosis was tested using mice deficient in LPS-binding protein (LBP-/-). LBP-/- mice had less liver injury and fibrosis (Siruis Red staining and hydroxyproline content) compared to wild type mice after bile duct ligation. In conclusion, these data demonstrate endotoxin in a CD14-dependent manner exacerbates hepatic fibrogenesis and macrophage activation to produce oxidants and cytokines following bile duct ligation.