BACKGROUND: Cholangiocarcinoma (CCA) is one of the most refractory malignancies with a grave prognosis. 2-Methoxy-4-vinylpenol (2M4VP) is a well-known flavonoid having beneficial functions in human health including an anti-cancer effect in various malignant tumours. However, the anti-cancer effect of 2M4VP against CCA remains ambiguous. PURPOSE: The present study aimed to investigate the bio-activity of 2M4VP in human CCA. METHODS: Biological activities of 2M4VP were examined using in vitro assays. Prognostic values were assessed using Kaplan-Meier and Liptak's z score analyses. RESULTS: 2M4VP significantly attenuated the anti-migratory features of CCA cells when compared to human intrahepatic bile duct epithelial cells (HIBEpiC), but not in viability. The anti-cancer effect of 2M4VP was induced by down-regulating the expression of cellular sonic hedgehog (Shh) signalling molecule such as GLI family zinc finger 3 (GLI3). In addition, 2M4VP treatment evoked a synergic anti-cancer effect when used in combination with gemcitabine. Furthermore, high GLI3 expression was significantly associated with a poor prognosis in patients with CCA. CONCLUSIONS: These results collectively indicate that 2M4VP might be a natural reagent against CCA metastasis, functioning through the ablating Shh signalling associated GLI3 expression, which provides the rationale for further investigation and a potential clinical trial using 2M4VP against CCA.