Lysine propionylation is a reversible and widely distributed post-translational modification that is known to play a regulatory role in both eukaryotes and prokaryotes. However, the extent and function of lysine propionylation in photosynthetic organisms remains unclear. Cyanobacteria are the most ancient group of Gram-negative bacteria capable of oxygenic photosynthesis, and are of great importance to global carbon and nitrogen cycles. Here, we carried out a systematic study of lysine propionylaiton in cyanobacteria where we used Synechocystis sp. PCC 6803 (Synechocystis) as a model. Combining high-affinity anti-propionyllysine pan antibodies with high-accuracy mass spectrometry (MS) analysis, we identified 111 unique lysine propionylation sites on 69 proteins in Synechocystis. Further bioinformatic analysis showed that a large fraction of the propionylated proteins were involved in photosynthesis and metabolism. The functional significance of lysine propionylation on the enzymatic activity of fructose-1,6-bisphosphatase (FbpI) was studied by site-directed mutagenesis and biochemical studies. Further functional studies revealed that the propionylation level of subunit II of photosystem I (PsaD) was obviously increased after high light (HL) treatment, suggesting that propionylation may be involved in high light adaption in Synechocystis. Thus, our findings provide novel insights into the range of functions regulated by propionylation and reveal that reversible propionylation is a functional modification with the potential to regulate photosynthesis and carbon metabolism in Synechocystis, as well as in other photosynthetic organisms.