Sirt5 is identified as an NAD-dependent protein lysine desuccinylase and demalonylase. Unlike other sirtuins, which primarily exhibit deacetylase activity, Sirt5 shows significant activity in deacylating lysine residues with succinyl and malonyl groups. This activity is explained by the presence of specific residues (Arg105 and Tyr102) in the acyl pocket of Sirt5. Mass spectrometry identified several mammalian proteins with succinyl or malonyl lysine modifications. Deletion of Sirt5 in mice increased the level of succinylation on carbamoyl phosphate synthase 1, a known target of Sirt5. These findings suggest that protein lysine succinylation and malonylation may be reversible posttranslational modifications regulated by Sirt5.Sirt5 is identified as an NAD-dependent protein lysine desuccinylase and demalonylase. Unlike other sirtuins, which primarily exhibit deacetylase activity, Sirt5 shows significant activity in deacylating lysine residues with succinyl and malonyl groups. This activity is explained by the presence of specific residues (Arg105 and Tyr102) in the acyl pocket of Sirt5. Mass spectrometry identified several mammalian proteins with succinyl or malonyl lysine modifications. Deletion of Sirt5 in mice increased the level of succinylation on carbamoyl phosphate synthase 1, a known target of Sirt5. These findings suggest that protein lysine succinylation and malonylation may be reversible posttranslational modifications regulated by Sirt5.