Abstract Recent studies revealed that mitochondrial Ca 2+ channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca 2+ uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca 2+ uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca 2+ overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca 2+ uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca 2+ uptake. Our data demonstrate that mitochondrial Ca 2+ uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca 2+ sensitivity. UCP2/3 normalize Ca 2+ sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca 2+ uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca 2+ uniporter by PRMT1 and UCP2/3.