Mitochondrial Ca²⁺ uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signals, to tune out small [Ca²⁺]_c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca²⁺ accumulation during small [Ca²⁺]_c elevations but an attenuated response to agonist-induced [Ca²⁺]_c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca²⁺]_c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca²⁺ binding, yet cells show impaired oxidative metabolism and sensitization to Ca²⁺ overload. Collectively, the data indicate that MICU1 senses the [Ca²⁺]_c to establish the uniporter's threshold and gain, thereby allowing mitochondria to properly decode different inputs.