Sirtuins are NAD⁺-dependent protein deacetylases and have been implicated in the regulation of metabolism, stress responses, and aging. Three sirtuins are located in mitochondria: SIRT3, 4, and 5. SIRT3 deacetylates and regulates the enzymatic activity of many metabolic enzymes in mitochondria, whereas SIRT5 removes two novel post-translational modifications, lysine malonylation and succinylation. Here, we review the current knowledge of how mitochondrial sirtuins function in metabolism and metabolic diseases, and offer a conceptual model how they may regulate mitochondrial function through distinct deacylation activities (deacetylation, demalonylation, or desuccinylation).