The study investigates the role of acetylcholine and its receptor, the alpha 5 nicotinic acetylcholine receptor subunit (CHRN5), in the progression of intrahepatic cholangiocarcinoma (ICC). Acetylcholine, derived from both nerves and tumor cells, promotes ICC metastasis by inducing epithelial-mesenchymal transition (EMT) and enhancing nerve infiltration. The acetylcholine/CHRN5 axis activates the GSK3β/β-catenin signaling pathway through Ca2+-mediated activation of Ca/calmodulin-dependent protein kinases (CAMKII), leading to increased β-catenin expression and BDNF secretion, which further promotes axonogenesis. The CAMKII inhibitor KN93 significantly inhibits the migration of ICC cells and enhances their sensitivity to gemcitabine, suggesting a potential therapeutic strategy for ICC metastasis. The study highlights the importance of targeting the acetylcholine/CHRN5 axis in the treatment of ICC.The study investigates the role of acetylcholine and its receptor, the alpha 5 nicotinic acetylcholine receptor subunit (CHRN5), in the progression of intrahepatic cholangiocarcinoma (ICC). Acetylcholine, derived from both nerves and tumor cells, promotes ICC metastasis by inducing epithelial-mesenchymal transition (EMT) and enhancing nerve infiltration. The acetylcholine/CHRN5 axis activates the GSK3β/β-catenin signaling pathway through Ca2+-mediated activation of Ca/calmodulin-dependent protein kinases (CAMKII), leading to increased β-catenin expression and BDNF secretion, which further promotes axonogenesis. The CAMKII inhibitor KN93 significantly inhibits the migration of ICC cells and enhances their sensitivity to gemcitabine, suggesting a potential therapeutic strategy for ICC metastasis. The study highlights the importance of targeting the acetylcholine/CHRN5 axis in the treatment of ICC.