Mitochondria take up Ca through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca signalling and cell death. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca transport. To prevent detrimental Ca overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca concentrations to switch MCU on and off. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca-activated states. These structures define the architecture of this multicomponent Ca-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca overload.