Biliary atresia (BA) is a congenital, progressive, fibro-obliterative disease of the extrahepatic biliary tree and the most common cause of end-stage liver disease in children. BA is characterized by extensive intrahepatic proliferating ductular reactions that may contribute to biliary fibrosis. Lineage tracing during experimental cholestasis indicates that cells within ductular reactions derive from PROM1-expressing hepatic progenitor cells. Given the role of Notch signaling in normal biliary development, we hypothesize that activated Notch signaling promotes the formation of ductular reactions in BA. Liver samples collected from BA infants at Kasai portoenterostomy and age-matched controls, as well as from wild-type and Prom1 knockout mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced experimental cholestasis were analyzed histologically using immunofluorescence and by quantitative polymerase chain reaction. Increased expression of genes encoding Notch ligand JAG1 and its receptor NOTCH2 was observed in BA livers compared with control by quantitative polymerase chain reaction analyses. Livers of DDC-treated mice, which exhibit cytokeratin-19-positive ductular reactions typical of BA livers, demonstrated significant increases in the expression level of the gene encoding Notch2, as well as downstream Notch target gene Hes1 compared with control. Prom1 knockout mice exhibit diminished ductular reactions and decreased levels of Jag1 and Hes1 compared with littermate controls. Human BA and cholestasis induced by DDC are associated with Notch signaling activation. Null mutation of Prom1 is associated with decreased ductular reactions and decreased Notch signaling activation during DDC treatment. These data are consistent with Notch signaling promoting ductular reactions of Prom1 expressing progenitor cells in BA.