Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte canalicular membrane, supposedly with its active site in the extracellular (luminal) mileau. To determine its function in biliary fluid and acid/base output, the common bile duct of anesthetized Car14-knockout (KO) and wild type (WT) mice aged 11, 20 and 52 weeks was cannulated, the gallbladder ligated, hepatic bile flow measured gravimetrically, and proton/HCO3- output by microtitration, before and during stimulation with intravenously applied taurocholic acid TCA. Systemic acid/base balance was controlled by tracheal intubation and artificial ventilation by isoflurane anesthesia, and by infusion of bicarbonate-containing Ringer’s solution into the carotis, and acid/base parameters determined in arterial blood obtained during the biliary drainage showed no significant differences between Car14 KO and WT mice. Morphological alterations and hepatic damage were assessed by studying gene and/or protein expression for proinflammatory cytokines, fibrosis and cholangiocyte proliferation markers, and by histochemical and immunohistochemical assessment, in Car14 KO and WT livers of mice aged 3, 6, 11, 20 and 52 weeks. Biliary basal, and more so TCA-stimulated HCO3- output was significantly reduced in Car14 KO mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Interestingly, Car14 KO mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein. The results suggest that the membrane-bound Car14 is essential to biliary HCO3- output, mixed micelle formation and bile acid neutralization, and that its loss results in gradual development of small bile duct disease and hepatic fibrosis. At young age, bile flow is not compromised, suggesting that Car14-knockout mice may be an optimal model to study the protective role of biliary canalicular HCO3- against luminal noxious substances to the cholangiocyte.