Purpose <p>Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis and complex tumour microenvironment. We previously demonstrated that doublecortin-like kinase 1 (DCLK1) is associated with epithelial–mesenchymal transition (EMT) in CCA; however, its biological functions and clinical relevance remain unclear. Herein, we elucidated the role of DCLK1 in CCA progression, focusing on its effects on angiogenesis, and evaluated its potential as a prognostic marker.</p> Methods <p>We analysed bulk and single-cell RNA sequencing data to characterise DCLK1 expression and function in CCA. Functional validation was performed using conditioned medium from DCLK1-modified CCA cells, as well as endothelial tube formation, migration, and invasion assays. Tumour vascular density was assessed using a xenograft model. We used a human angiogenesis-related antibody array, western blotting, quantitative real-time PCR, and nuclear translocation assays to examine CXCL5 expression and NF-κB pathway activity.</p> Result <p>We found that DCLK1 was upregulated in CCA and correlated with advanced disease and poor survival. DCLK1⁺ malignant cells displayed pro-angiogenic features and increased paracrine interaction with endothelial cells. Conditioned medium from DCLK1-overexpressing tumour cells enhanced endothelial tube formation, migration, and invasion, whereas DCLK1 knockout reversed these effects. DCLK1-expressing tumours showed increased vascular density in vivo. Mechanistically, an angiogenesis antibody array and western blotting demonstrated that DCLK1 activated the NF-κB pathway to upregulate CXCL5 expression. Pharmacological inhibition of NF-κB or neutralization of CXCL5 significantly attenuated DCLK1-related pro-angiogenic responses in vitro.</p> Conclusion <p>DCLK1 activated the NF-κB pathway to upregulate CXCL5 secretion; inhibiting NF-κB or CXCL5 reduced DCLK1-related pro-angiogenic capacity.</p>