As a novel protein post-translational modification, lysine succinylation is widely involved in metabolism regulation. In this study, we focused on the distribution of lysine succinylation sites and their physiological functions in Saccharopolyspora erythraea. Using high-resolution 4D label free mass spectrometry, a large and global protein succinylome was identified in a hypersuccinylated strain E3ΔsucC. The results showed that succinylated proteins are predominantly involved in protein synthesis-related pathways (e.g., ribosomes, tRNA) and metabolic pathways, such as the TCA cycle. Proteins in these pathways generally have a higher lysine content, suggesting that lysine succinylation may have a greater regulatory role in biochemical reactions involving acidic substrates. Motif analysis revealed that charged amino acids (D, E, K, R and W) display a more regular distribution around acylation sites, implying that the polar effect between residues may be the key factor influencing lysine succinylation. Based on predicted protein structures, we highlighted the potential impact of lysine succinylation on enzyme activity in the TCA cycle. In conclusion, this study offers valuable insights into the regulation of lysine succinylation and contributes to a comprehensive understanding of its physiological functions in actinomyces.