Mitochondrial calcium uptake plays a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients, and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here, we utilize an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics, and organelle proteomics. RNA interference against 13 top candidates highlighted one gene that we now call mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the organelle’s inner membrane and has two canonical EF hands that are essential for its activity, suggesting a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high capacity mitochondrial calcium entry. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.