Mitochondrial Ca²⁺ (Ca²⁺_m) uptake is mediated by an inner membrane Ca²⁺ channel called the uniporter. Ca²⁺ uptake is driven by the considerable voltage present across the inner membrane (ΔΨ_m) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca²⁺ concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca²⁺]_m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca²⁺, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca²⁺ threshold for Ca²⁺_m uptake without affecting the kinetic properties of MCU-mediated Ca²⁺ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca²⁺_m uptake that is essential to prevent [Ca²⁺]_m overload and associated stress.