The mitochondrial uniporter (MCU) is an ion channel that mediates Ca²⁺ uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca²⁺ signaling. Matrix Ca²⁺ concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca²⁺ overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca²⁺ concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca²⁺ inhibition of MCU Ca²⁺ currents, resulting in MCU channel activation, enhanced mitochondrial Ca²⁺ uptake, and constitutively elevated matrix Ca²⁺ concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca²⁺. Thus, mitochondria are protected from Ca²⁺ depletion and Ca²⁺ overload by a unique molecular complex that involves Ca²⁺ sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.