Updated publication reference for PubMed record(s): 31337674. As a recently discovered protein posttranslational modification in eukaryotes, lysine succinylation has attracted increasing interest due to its ability to regulate several critical cellular processes including catabolism, β-oxidation, and ketogenesis. Nevertheless, understanding of the regulation mechanism is still at an early stage due to the lack of identified specific desuccinylases in most microorganisms.Here, in the model soil bacteria Streptomyces coelicolor, we biochemically characterized a sirtuin-like protein ScCobB2 as a divergent desuccinylase. Based on it, we identified a total of 673 unique succinylated sites, of which 470 sites in 317 proteins were quantified by combining tandem mass tag labeling, antibody affinity enrichment, and LC−MS/MS analysis. Furtheranalyses of the quantitative succinylome revealed that at least 114 proteins representing two major pathways, carbon metabolism and protein biosynthesis, are obviously hypersuccinylated in ∆ScCobB2 cells. We experimentally examined the regulatory roles of ScCobB2 on 13 hypersuccinylated proteins, including glyceraldehyde-3-phosphate dehydrogenase, aconitate hydratase, and several ribosomal proteins, the results of which suggested a high confidence in our quantitative data. This work provided the first discovery of a specificdesuccinylase in bacteria, and demonstrated it has pivotal regulatory role in multiple biological processes for S. coelicolor, laid the foundation for future research of succinylation regulation in other microorganisms.