The activation of Ephrin (Eph) receptors, the largest tyrosine kinase families of cell surface receptor, has recently been addressed in human cholangiocarcinoma (CCA). Therefore, the present study aimed to investigate the role of Eph receptors and its ligands in CCA. Of all 50 cases of human CCA tested, immunohistochemical staining demonstrated that EphB2, EphB4, ephrinB1, and ephrinB2 were 100 % positive in CCA tissues with overexpressions of the above proteins as 56, 56, 70, and 48 % of cases, respectively. High expression of EphB2 was significantly correlated with the metastatic status of patients (P = 0.027). We also found that the high co-expression level of EphB2/ephrinB1 or EphB2/ephrinB2 were significantly correlated with the metastatic status of the patients (P = 0.034 and P = 0.024). Furthermore, we showed that the high co-expression level of EphB4/MVD and ephrinB1/MVD were significantly correlated with the metastasis status of CCA patients (P = 0.012 and P = 0.029). We further demonstrated that the EphB2 suppression using siRNA significantly reduced CCA cell migration by decreasing the phosphorylation of focal adhesion kinase (FAK) and paxillin. In conclusion, the upregulation of EphB2 receptors and its specific ligands (ephrinB1 and ephrinB2) leads to CCA metastasis. Suppression of EphB2 expression as well as inhibition of its downstream signaling proteins might serve as possible therapeutic strategies in human CCA.