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 regulatory mechanisms is still at an early stage due to the lack of identified specific desuccinylases in microorganisms. Here, in the model soil bacterium Streptomyces coelicolor, we biochemically characterized a sirtuin-like protein ScCobB2 as a divergent desuccinylase. Based on it, we were able to identify a total of 673 unique succinylated sites, of which 470 sites in 317 proteins were quantified by comparing the ΔScCobB2 to the wild-type succinylome via LC-MS/MS analysis. Further analyses of the quantitative succinylome revealed that at least 114 proteins representing two major pathways, protein biosynthesis and carbon metabolism, are obviously hypersuccinylated in ΔScCobB2 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 specific desuccinylase in bacteria and demonstrated it has pivotal regulatory roles in multiple biological processes of S. coelicolor, laying the foundation for future research of succinylation regulation in other microorganisms.