Protein acylation plays important roles in bacterial pathogenesis through regulation of enzymatic activity, protein stability, nucleic acid binding ability, and protein-protein interactions. Mycobacteria, a genus including invasive pathogens known to cause serious diseases, shapes its pathogenicity through adaptation of its energy metabolism to microenvironments encountered within mammalian hosts. In this process, acetyl-CoA and propionyl-CoA function as important intermediates. However, the function of acetyl-CoA/propionyl-CoA driven protein acylation remains to be elucidated. Herein, we systematically investigated protein acetylome/propionylome in the nonpathogenic Mycobacterium smegmatis through antibody-enrichment-based proteomic analysis in which 146 acetylated sites on 121 proteins and 26 propionylated sites on 25 proteins were identified. After that, characteristic differences of the two acylomes were elucidated through such bioinformatic methods as motif analysis, protein-protein analysis, Gene Ontology analysis, and KEGG analysis. In addition, quantitative mass spectrometric method was used to evaluate the site-specific and motif-biased catalytic mechanism mediated by the cAMP-dependent acetyltransferase MsKat in M. smegmatis. Furthermore, we raised the possibility that both O-serine and Nε-lysine acetylation might coregulate the propionyl-CoA synthetase. This study described the landscape of acetylome and propionylome in the M. smegmatis, showing an unexpected role of protein acylation regulation in mycobacteria.