Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca²⁺ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca²⁺, the possibility that physiological stimuli that raise the [Ca²⁺] of the cytoplasm could trigger major mitochondrial Ca²⁺ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca²⁺] rises. The direct measurement of mitochondrial [Ca²⁺] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca²⁺ signal is always paralleled by a much larger rise in [Ca²⁺] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca²⁺ transport and novel, unexpected results have altered long‐standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca²⁺ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca²⁺ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca²⁺ sources; (iii) the physiological consequences of Ca²⁺ transport for both mitochondrial function and the modulation of the cytoplasmic Ca²⁺ signal; and (iv) evidence that alterations of mitochondrial Ca²⁺ signalling may occur in pathophysiological conditions.