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All living beings require energy and the ultimate source for this energy in eukaryotes are mitochondria – originally an organelle of bacterial origin and eventually incorporated into the cytoplasm. Mitochondrial bioenergetics and dynamics are essential for the maintenance of cellular and metabolic homeostasis and any imbalance can result in diseases such as cancer. The mitochondrial matrix is a vital site for many enzymes responsible for mitochondrial function. Three members of the sirtuin family (SIRT3, SIRT4 and SIRT5) localize into the mitochondrial matrix and are, like all sirtuins, dependent on NAD+. They regulate various cellular processes such as cell cycle, gene expression, energy homeostasis and post translational processes, for example deacetylation, ADP-ribosylation, etc. Studies so far suggest that mitochondrial sirtuins (mtSirts) can act both as tumor promotor and suppressor, and therefore play contrasting roles in various cancers.