Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD+-dependent lysine deacylase and removes both succinyl and malonyl group. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild type (WT) and Sirt5-/- mice. 1137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in the Sirt5-/- animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5-/- compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison of our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.