Background <p>Cancer-associated fibroblasts (CAFs), integral components of the tumor microenvironment, substantially influence cholangiocarcinoma (CCA) progression and clinical outcomes. This study aimed to identify CAF-related genes implicated in CCA pathogenesis.</p> Methods <p>Differentially expressed genes (DEGs) between CCA and non-tumorous samples were extracted from the Gene Expression Omnibus GSE107943 dataset, then intersected with cancer-associated fibroblast signature genes (CAFSGs) to derive DE-CAFSGs. Mendelian randomization (MR) analysis was subsequently conducted to determine causally linked biomarkers. The diagnostic efficacy of these biomarkers was evaluated via receiver operating characteristic (ROC) curve analysis using data from GSE107943 and the TCGA-CHOL cohort. Functional annotation of representative DE-CAFSGs was performed through single-gene gene set enrichment analysis. The expression trends of these biomarkers were preliminarily explored using RT-PCR, Western blotting, and immunohistochemistry.</p> Results <p>A total of 135 DE-CAFSGs were identified, among which GRN, PLXNC1, and ORMDL3 emerged as CCA-associated biomarkers. GRN and PLXNC1 were identified as risk-associated, while ORMDL3 exhibited a protective association. Each demonstrated robust discriminatory capacity, with area under the ROC curve values exceeding 0.8 in both datasets. Enrichment analysis revealed significant associations with pathways including retinol metabolism, cytochrome P450-mediated drug metabolism, and the complement and coagulation cascades.</p> Conclusion <p>GRN, PLXNC1, and ORMDL3 represent key CAF-associated genes in CCA, shedding light on potential molecular mechanisms of disease development.</p>