In the heart, augmented Ca²⁺ fluxing drives contractility and ATP generation through mitochondrial Ca²⁺ loading. Pathologic mitochondrial Ca²⁺ overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca²⁺ uptake is primarily mediated by the mitochondrial Ca²⁺ uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca²⁺ uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca²⁺ challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca²⁺ levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca²⁺ after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca²⁺ loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.