ABSTRACT Post-translational modifications (PTMs) in Staphylococcus aureus play crucial roles in regulating diverse biological processes, exerting a substantial impact on protein functions within this pathogen. In this study, we comprehensively analyzed the overall patterns of three lysine acylation types, identifying 1,249 acetylated sites, 871 succinylated sites and 67 malonylated sites in S. aureus. Further bioinformatic analysis showed that the lysine acetylation and succinylation had a preference for glutamate surrounding the modified lysine residues. Pathway enrichment showed that modified substrates were associated with ribosomes and metabolic functions. Additional functional exploration showed that the lysine succinylation affected the enzymatic activity of glutamyl-tRNA amimide transferases and carbamoyl phosphate synthases were linked to their enzymatic activities. In conclusion, our study enhanced the comprehension of lysine acylations in S. aureus and identified potential targets related with its pathogenicity at PTM level.