Abstract Background Cholestatic-fibrotic liver diseases have a high unmet medical need. Tight junctions, which maintain the blood-biliary-barrier in the liver, have important biological functions that impact cholestatic diseases. A main component of the hepatic tight junction is claudin-3. Aims In this study, we explored the function of claudin-3 and demonstrate its potential as a new drug target in cholestatic-fibrotic liver diseases. Methods Adult C57BL/6 N background Cldn3+/+ or Cldn3-/- mice were subjected to extra- and intrahepatic cholestasis via surgical ligation of the common bile duct (BDL) or chemically, by gavage or feeding alpha-naphthyl isothiocyanate (ANIT). Hepatic necrosis and serum liver injury markers were determined. Bile flow and bicarbonate levels were quantified. Bile acid levels were measured by LC-MS/MS in serum and liver tissue. Claudin-3 was knocked down using liver specific claudin-3 GalNAc-siRNAs. Results Claudin-3 is expressed in normal- and cholestatic liver tissues. Cldn3-/- mice have a higher bile-flow rate, more bicarbonate secretion, and the bile is less concentrated with bile acids and bilirubin. Following bile duct ligation or ANIT, Cldn3-/- mice showed macroscopically less signs of bile acid retention and a different bile color. Hepatic bile acid levels were significantly lower in normal- or cholestasis challenged Cldn3-/- mice. Following BDL, ANIT gavage- or ANIT feeding, liver injury in was remarkably ameliorated in Cldn3-/- mice. Liver specific knockdown of claudin-3 using GalNAc-siRNAs ameliorated cholestasis at a lower success rate compared to the complete knockout. Conclusion Knockout or GalNAc-siRNA mediated inhibition of claudin-3 protected the liver from injury in models of extra- or intrahepatic cholestasis. Further pre-clinical and clinical studies need to be conducted to show whether claudin-3 inhibition is an efficient and safe therapeutic approach for the treatment of cholestatic liver diseases. As next step towards translation of our results, we are currently screening and optimizing human GalNAc conjugated claudin-3 inhibitors.