Mitochondrial Ca²⁺ homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca²⁺ channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca²⁺ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca²⁺ uptake. MCU overexpression doubled the matrix Ca²⁺ concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca²⁺ concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca²⁺ uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.