Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2'-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation. Here, we review and discuss recent progress in the study of mitochondrial sirtuins and their targets.