Calcium ions (Ca²⁺) are some of the most versatile signalling molecules, and they have many physiological functions, prominently including muscle contraction, neuronal excitability, cell migration and cell growth. By sequestering and releasing Ca²⁺, mitochondria serve as important regulators of cellular Ca²⁺. Mitochondrial Ca²⁺ also has other important functions, such as regulation of mitochondrial metabolism, ATP production and cell death. In recent years, identification of the molecular machinery regulating mitochondrial Ca²⁺ accumulation and efflux has expanded the number of (patho)physiological conditions that rely on mitochondrial Ca²⁺ homeostasis. Thus, expanding the understanding of the mechanisms of mitochondrial Ca²⁺ regulation and function in different cell types is an important task in biomedical research, which offers the possibility of targeting mitochondrial Ca²⁺ machinery for the treatment of several disorders.