The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Inhibition of mitochondrial Ca2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo, and slower cell proliferation in vitro associated with delayed progression through S phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca2+ content and membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis, glutaminolysis, sensitized to glucose and glutamine limitation, and altered agonist-induced cytoplasmic and mitochondrial Ca2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca2+ uptake for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.