The contribution that mitochondrial bioenergetics could have in cancer development is debated. Here, we have generated HCT116-derived colocarcinoma cell lines expressing different levels of the beta catalytic subunit of the mitochondrial H+-adenosine triphosphate synthase to assess the contribution of mitochondrial bioenergetics in colon cancer progression. The generated cells exhibit large ultrastructural, transcriptomic, proteomic and functional differences in their mitochondria and in their in vivo tumor forming capacity. We show that the activity of oxidative phosphorylation defines the rate of glucose utilization by aerobic glycolysis. The aggressive cellular phenotype, which is highly glycolytic, is bound to the deregulated expression of genes involved in metabolic processes, the regulation of the cell cycle, apoptosis, angiogenesis and cell adhesion. Remarkably, the molecular and ultrastructural analysis of the tumors derived from the three HCT116 cell lines under study highlight that tumor promotion inevitably requires the selection of cancer cells with a repressed biogenesis and functional activity of mitochondria, i.e. the highly glycolytic phenotype is selected for tumor development. The tumor forming potential of the cells is a non-genetically acquired condition that provides the cancer cell with a cell-death resistant phenotype. An abrogated mitochondrial respiration contributes to a diminished potential for reactive oxygen species signaling in response to 5-fluorouracil treatment. Treatment of cancer cells with dichloroacetate partially restores the functional differentiation of mitochondria and promotes tumor regression, emphasizing the reversible nature of the metabolic trait of cancer.