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. Display omitted •The inner membrane mitochondrial MCU ion channel mediates Ca2+ uptake into the matrix•MCU channel activity is governed by matrix Ca2+ concentration through EMRE•EMRE-dependent regulation requires MICU1, MICU2, and cytoplasmic Ca2+•EMRE couples Ca2+ sensors on both sides of the inner membrane to regulate MCU Using patch clamp electrophysiology, Vais et al. demonstrate that mitochondrial matrix Ca2+ concentration regulates the activity of MCU, the major mitochondrial Ca2+ influx pathway. Mitochondria are protected from Ca2+ depletion and overload by a unique complex involving Ca2+ sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.