Populations of tumor cells display a remarkable diversity in their phenotypic traits, including their drug resistance and ability to form metastases. This heterogeneity has recently been explained by the cancer stem cell (CSC) model. In this study, we investigated the presence of CSCs in established malignant mesothelioma (MM) cell lines and characterized their molecular and metabolic features. Mesothelioma cells successfully formed mesospheres as a model of cancer stem cells. Mesospheres had the capacity to initiate tumours in immunodeficient mice and maintained this capacity through serial transplantation. We identified CD24, ABCG2 and Oct4 as potential markers of mesothelioma stem cells and showed that CD24 knockdown cells exhibit loss of sphere formation capacity, change of morphology, decrease in the rate of proliferation and lower tumorigenicity. Subjecting mesospheres to serial transplantation resulted in more aggressive tumors, which expressed consistently higher levels of stem cell markers. CSCs have distinct molecular and biological signature, but little is known about their mitochondrial and metabolic features. Our data reveal that like somatic stem cells, MM CSCs exhibit lower levels of normalised oxidative phosphorylation and mtDNA, and use glycolysis as their main source of energy production, thus have increased levels of lactate formation and glucose uptake and lower levels of ATP and mitochondrial proteins. They also feature enhanced mitochondrial membrane potential [...] and ROS generation.