Mitochondria are now recognized as one of the main intracellular calcium-storing organelles which play a key role in the intracellular calcium signalling. Indeed, besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca2+) transients, thus controlling cytosolic Ca2+ signals and Ca2+-dependent protein activity. It has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is a ubiquitous phenomenon described in every cell system in which the issue has been addressed. In this chapter, we will review the molecular mechanisms involved in the regulation of mitochondrial calcium cycling in physiological conditions with particular regard to the role played by the mitochondrial Na+/Ca2+ exchanger.