Lithocholate-3-O-glucuronide is a cholestatic agent. Herein, the effect of lithocholate-3-O-glucuronide on biliary glutathione excretion, which has been suggested as a driving force for the bile acid independent canalicular bile flow, was examined in rats. Intravenously injected lithocholate-3-O-glucuronide (1.6 μmol/100 g body wt.) caused a transitional decrease in bile flow (67% of the basal value) and depressed biliary glutathione excretion to 15% of the basal value, which corresponded to biliary lithocholate-3-O-glucuronide excretion. A lower dose of lithocholate-3-O-glucuronide (1.0 μmol/100 g body wt.) also inhibited biliary glutathione excretion (54%) with a slight decrease in bile flow (80%). These findings indicate that a decrease in bile acid independent bile flow due to the inhibition of biliary glutathione excretion is one of the initiating factors to cause cholestasis induced by lithocholate-3-O-glucuronide.