Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu−/− mice display no overt baseline phenotype and are protected against mCa2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu−/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metabolism during acute stress. Display omitted •The MCU is dispensable for baseline homeostatic cardiac function•Deletion of Mcu protects against myocardial IR injury by reducing MPTP activation•The MCU is required to match energetics with contractile demand during stress•A slow MCU-independent uptake mechanism may maintain basal matrix mCa2+ content Luongo et al. show, using a conditional knockout mouse model, that the mitochondrial Ca2+ uniporter (MCU), although dispensable for homeostatic function, is necessary for the cardiac “fight-or-flight” contractile response and a significant contributor to mitochondrial permeability transition during ischemia-reperfusion injury.