Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU^−/− mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU^−/− mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU^−/− mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU^−/− mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not seem to protect MCU^−/− cells and tissues from cell death, although MCU^−/− hearts fail to respond to the PTP inhibitor cyclosporin A. Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.