Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease. Display omitted •Human biliary epithelial cells (hBECs) can be isolated from discarded human livers•hBECs show regenerative properties when grafted into a biliary disease mouse model•Mice transplanted with hBECs regenerate bile ducts and show improved liver function•hBECs can be cultured in good manufacturing process conditions for clinical use In this manuscript, Forbes and colleagues isolate and expand human biliary epithelial cells (hBECs) from discarded livers. Upon transplantation into a mouse model of biliary disease, hBECs regenerate and repair damaged bile ducts, offering a potential therapy for biliary disease.