A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result intumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential tounderstand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternativestrategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in orderto point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cellswere compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identifya metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift frommitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic dataprovide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactatedehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, weshow that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone andshows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis maybe a potentially effective strategy to target BCSCs.