Alpha5 nicotinic acetylcholine receptor (CHRNA5) promotes metastasis of intrahepatic cholangiocarcinoma (ICC) by activating acetylcholine signaling. The study shows that ICC cells and infiltrating nerves generate a microenvironment rich in acetylcholine, which induces epithelial-mesenchymal transition (EMT) and promotes ICC metastasis. Acetylcholine interacts with CHRNA5 to activate the Ca/calmodulin-dependent protein kinase II (CAMKII) pathway, which in turn activates GSK3β and stabilizes β-catenin. This pathway enhances ICC migration and resistance to gemcitabine. The acetylcholine/CHRNA5 axis also increases brain-derived neurotrophic factor (BDNF) expression, which promotes axonogenesis and forms a feedback loop between ICC cells and nerves. The CAMKII inhibitor KN93 significantly inhibits ICC migration and enhances gemcitabine sensitivity. These findings suggest that targeting the acetylcholine/CHRNA5 axis could be a novel therapeutic strategy for ICC. The study also highlights the role of nerves in ICC progression through acetylcholine signaling and the importance of β-catenin in regulating ICC metastasis and chemotherapy resistance. The research provides new insights into the mechanisms underlying ICC progression and offers potential therapeutic targets for ICC treatment.Alpha5 nicotinic acetylcholine receptor (CHRNA5) promotes metastasis of intrahepatic cholangiocarcinoma (ICC) by activating acetylcholine signaling. The study shows that ICC cells and infiltrating nerves generate a microenvironment rich in acetylcholine, which induces epithelial-mesenchymal transition (EMT) and promotes ICC metastasis. Acetylcholine interacts with CHRNA5 to activate the Ca/calmodulin-dependent protein kinase II (CAMKII) pathway, which in turn activates GSK3β and stabilizes β-catenin. This pathway enhances ICC migration and resistance to gemcitabine. The acetylcholine/CHRNA5 axis also increases brain-derived neurotrophic factor (BDNF) expression, which promotes axonogenesis and forms a feedback loop between ICC cells and nerves. The CAMKII inhibitor KN93 significantly inhibits ICC migration and enhances gemcitabine sensitivity. These findings suggest that targeting the acetylcholine/CHRNA5 axis could be a novel therapeutic strategy for ICC. The study also highlights the role of nerves in ICC progression through acetylcholine signaling and the importance of β-catenin in regulating ICC metastasis and chemotherapy resistance. The research provides new insights into the mechanisms underlying ICC progression and offers potential therapeutic targets for ICC treatment.