Background <p>Cholangiocarcinoma (CCA) remains a challenging malignancy with limited therapeutic options and poor prognosis. This study investigates the role of glycerol-3-phosphate dehydrogenase 2 (GPD2) and associated glycolysis-related genes in cancer, with a focus on CCA.</p> Methods <p>We employed a comprehensive multi-omics approach, including genomic, transcriptomic, and single-cell analyses, to explore GPD2’s function across various cancers. In vitro experiments validated GPD2’s role in CCA. A glycolysis-related prognostic signature was developed and analyzed for its clinical and biological implications.</p> Results <p>GPD2 was overexpressed in most cancers and correlated with adverse outcomes, genomic instability, and altered immune infiltration. In CCA, GPD2 knockdown suppressed cell proliferation, migration, and glycolytic activity. A four-gene glycolysis-related signature (PTBP3, REEP3, GPRC5A, HN1) successfully classified CCA patients into low and high-risk categories, demonstrating strong prognostic value. The high-risk group displayed distinct immune infiltration patterns, characterized by a decrease in CD8 + T cells and an elevation in M0 macrophages and neutrophils. Single-cell analysis showed that the signature genes were primarily expressed in malignant epithelial cells and provided clues into potential mechanisms of tumor-immune interactions.</p> Conclusion <p>This study highlights the potential of GPD2 and the glycolysis-related gene signature as prognostic biomarkers and therapeutic targets in CCA. The signature offers insights into tumor biology, immune interactions, and potential personalized treatment strategies, paving the way for improved management of this aggressive cancer.</p> Graphical Abstract <p></p>