Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca²⁺ concentrations, preventing deleterious Ca²⁺ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca²⁺] in situ and allowing tight physiological control. At low [Ca²⁺], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca²⁺], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca²⁺ signals generated in the cytoplasm.