Activation of developmental pathways has been recognized as a key mechanism for tumourigenesis and, hence, might be a valuable target for otherwise difficult to treat tumour entities such as biliary tract cancer (BTC). Therefore, we performed a comprehensive analysis of the Wnt signalling pathway in 9 BTC cell lines on cell blocks, xenograft tumours and on human tissue microarrays by real-time reverse transcription PCR and by immunochemistry. Furthermore, the effects of pharmacological pathway inhibition were investigated. As a result we found a significant positive correlation of Wnt pathway activation with cyclin D1 expression and the proliferation parameters Ki67, cell cycle distribution, and growth kinetics as well as the mesenchymal marker vimentin and an inverse correlation with E-cadherin in BTC cell lines in vitro and in vivo. In human BTC samples loss of membranous beta-catenin, an indicator of active Wnt signalling, correlated with vimentin expression and advanced tumour stage or metastasis, whereas membranous localisation of beta-catenin was associated with the differentiation marker cytokeratin-8/18 and differentiated tumour morphology (ductal or mixed type BTC). In addition, Wnt pathway inhibition by DMAT effectively reduced viability in all cancer cell lines, most effectively in those showing cytoplasmatic beta-catenin localisation, i.e. active Wnt signalling. In summary, activation of the Wnt pathway is associated with high proliferation, dedifferentiation and a solid morphology in human biliary tract cancer cell lines both in vitro and in vivo, and in human BTC tissues. Further investigation of the mechanism(s) of Wnt pathway activation and its inhibition may provide new molecular treatment strategies for biliary tract cancer.