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N^ε‐lysine acetylation, a reversible and highly regulated PTM, has been shown to occur in the model Gram‐negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram‐positive bacterium. Through anti‐acetyllysine antibody‐based immunoseparation of acetylpeptides followed by nano‐HPLC/MS/MS analysis, we identified 332 unique lysine‐acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. Fifity‐nine of the lysine‐acetylated proteins showed homology with lysine‐acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH, and YfmT, indicating that lysine acetylation …