Cholangiocarcinoma (CCA) is a devastating liver tumour arising from malignant transformation of bile duct epithelial cells. Cancer stem cells (CSC) are a subset of tumour cells endowed with stem-like properties, which play a role in tumour initiation, recurrence and metastasis. In appropriate conditions, CSC form 3D spheres (SPH), which retain stem-like tumour-initiating features. Here, we found different expression of iron proteins indicating increased iron content, oxidative stress and higher expression of CSC markers in CCA-SPH compared to tumour cells growing as monolayers. Exposure to the iron chelator desferrioxamine decreased SPH forming efficiency and the expression of CSC markers and stem-like genes, whereas iron had an opposite effect. Microarray profiles in CCA samples (n = 104) showed decreased H ferritin, hepcidin and ferroportin expression in tumours respect to surrounding liver, whereas transferrin receptor was up-regulated. Moreover, we found a trend toward poorer outcome in CCA patients with elevated expression of ferritin and hepcidin, two major proteins of iron metabolism. These findings, which represent the first evidence of a role for iron in the stem cell compartment as a novel metabolic factor involved in CCA growth, may have implications for a better therapeutic approach.