Advancing age is the major risk factor for the development of chronic diseases and is accompanied by 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 of 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.