Intra-tumoral hypoxia is an environment that promotes tumor cell migration, angiogenesis and epithelial- mesenchymal transition that accounts for a major mechanism of metastasis. Chloroquine potentially offers a new therapeutic approach with an 'old' drug for effective and safe cancer therapies, as it exerts anti-metastatic activity. We investigated the inhibitory effect of chloroquine on cholangiocarcinoma (CCA) cell migration under cobalt chloride (CoCl2)-stimulated hypoxia. We showed that chloroquine suppressed CCA cell migration under hypoxic-mimicking conditions on exposure to 100 μM CoCl2. Moreover, chloroquine stabilized the protein level of prolyl hydroxylase domain proteins (PHD-2) but reduced the levels of hypoxic responsive proteins such as hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF). It also suppressed epithelial mesenchymal transition (EMT) by increasing the ratio of E-cadherin to N-cadherin under hypoxic conditions. In conclusion, chloroquine can inhibit hypoxia-stimulated metastasis via HIF-1α/VEGF/EMT which may serve as a useful additional strategy for CCA therapy.