Protein acetylation is one of the post-translational modifications (PTMs) involved in regulating a myriad of cellular processes in bacteria. Increasing evidence demonstrates that lysine acetylation is involved in Mycobacterium tuberculosis (Mtb) virulence and pathogenesis. However, previous reports have detected acetylation at lysine residues using only reference strains. Here, we analyzed the global Nε- and O-acetylation of Mtb lineage 7 clinical isolates and H37Rv. Quantitative acetylome analysis resulted in identification of 2577 class-I acetylation sites derived from 987 proteins. These proteins were found to be involved in central metabolism, translation, stress responses and drug resistance. Interestingly, 261 acetylation sites on 164 proteins were differentially regulated between the two virulent strains. A total of 257 acetylation sites on 160 proteins were hypoacetylated in lineage 7. These proteins are involved in Mtb growth, virulence, energy metabolism, host-pathogen interaction and stress responses. Furthermore, Gene Ontology (GO) analysis of exclusively acetylated proteins identified revealed strain-specific enrichment of selected biological processes. Taken together, this study provides the first global analysis of O-acetylated proteins in Mtb. This quantitative acetylome data presents the abundance and diversity of acetylated proteins in Mtb and opens a new avenue of research in exploring the role of protein acetylation in fine-tuning Mtb physiology.