Advancing age is the major risk factor for the development of chronic diseases and is accompanied with changes in metabolic processes and mitochondrial dysfunction. Mitochondrial sirtuins (SIRT3-5) are part of the sirtuin family of NAD +-dependent deacylases and ADP-ribosyl transferases. The dependence on NAD + links sirtuin enzymatic activity to the metabolic state of the cell, poising them as stress sensors. Recent insights have revealed that SIRT3-5 orchestrate stress responses through coordinated regulation of substrate clusters rather than a few key metabolic enzymes. Additionally, mitochondrial sirtuin function has been implicated in the protection against age-related pathologies including neurodegeneration, cardiopathologies and insulin resistance. In this review, we highlight the molecular targets of SIRT3-5 and discuss their involvement in aging and age-related pathologies.