N-epsilon -Acetylation of lysine residues represents a frequently occurring post-translational modification widespread in bacteria that plays vital roles in regulating bacterial physiology and metabolism. However, the role of lysine acetylation in cyanobacteria remains unclear, presenting a hurdle to in-depth functional study of this post-translational modification. Here, we report the lysine acetylome of Synechococcus sp. PCC 7002 (hereafter Synechococcus) using peptide prefractionation, immunoaffinity enrichment, and coupling with high-precision liquid chromatographytandem mass spectrometry analysis. Proteomic analysis of Synechococcus identified 1653 acetylation sites on 802 acetylproteins involved in a broad range of biological processes. Interestingly, the lysine acetylated proteins were enriched for proteins involved in photosynthesis, for example. Functional studies of the photosystem II manganese- stabilizing protein were performed by site-directed mutagenesis and mutants mimicking either constitutively acetylated (K99Q, K190Q, and K219Q) or nonacetylated states (K99R, K190R, and K219R) were constructed. Mutation of the K190 acetylation site resulted in a distinguishable phenotype. Compared with the K190R mutant, the K190Q mutant exhibited a decreased oxygen evolution rate and an enhanced cyclic electron transport rate in vivo. Our findings provide new insight into the molecular mechanisms of lysine acetylation that involved in the negative regulation of oxygen evolution in Synechococcus and creates opportunities for in-depth elucidation of the physiological role of protein acetylation in photosynthesis in cyanobacteria.