SIRT3 is a soluble protein located in the mitochondrial matrix, and contains a mitochondrial processing peptide at the N-terminus. A set of crystal structures of human SIRT3 have been solved, including an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide and a structure with the dethioacetylated peptide bound. These structures show the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, a binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, prior to NAD+.