Lysine succinylation is a newly-identified protein post-translational modification pathway, which is present in both eukaryotic and prokaryotic cells, but its extent and function in photosynthetic organisms remain unexplored. We performed a global succinylome analysis of a model cyanobacterium Synechococcus sp. PCC 7002 using high-accuracy tandem mass spectrometry (MS/MS) analysis in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, we identified 265 lysine succinylation sites on 129 proteins, which were involved in various biological processes; however, a large proportion of the succinylation sites were present on proteins in the photosynthetic pathway. Functional studies showed that succinylation of the manganese-stabilizing protein PsbO can decrease the oxygen evolution rates of the photosystem (PS) II center and the efficiency of energy transfer during the photosynthetic reaction. Our results demonstrate that succinylation may be a mechanism involved in acclimation to high light in Synechococcus as well as play a regulatory role in photosynthesis. Molecular dynamics simulations elucidated a mechanism that may allow succinylation to influence the efficiency of photosynthesis by altering the conformation of PsbO, thereby hindering the interaction between PsbO and the PSII core. Our findings provide novel insights into the functions of succinylation in the regulation of photosynthesis, thereby suggesting that reversible succinylation may be an important regulatory mechanism during photosynthesis in Synechococcus, as well as in other photosynthetic organisms.