Calcium (Ca 2+ ) signalling acts a pleiotropic message within the cell that is decoded by the mitochondria through a sophisticated ion channel known as the Mitochondrial Ca 2+ Uniporter (MCU) complex. Under physiological conditions, mitochondrial Ca 2+ signalling is crucial for coordinating cell activation with energy production. Conversely, in pathological scenarios, it can determine the fine balance between cell survival and death. Over the last decade, significant progress has been made in understanding the molecular bases of mitochondrial Ca 2+ signalling. This began with the elucidation of the MCU channel components and extended to the elucidation of the mechanisms that regulate its activity. Additionally, increasing evidence suggests molecular mechanisms allowing tissue-specific modulation of the MCU complex, tailoring channel activity to the specific needs of different tissues or cell types. This review aims to explore the latest evidence elucidating the regulation of the MCU complex, the molecular factors controlling the tissue-specific properties of the channel, and the physiological and pathological implications of mitochondrial Ca 2+ signalling in different tissues.