Lysine malonylation (Kmal) is an evolutionarily conserved post-translational modification (PTM) that has been demonstrated to be involved in cellular and organismal metabolism. However, the role that Kmal plays in response to drought stress of the terrestrial cyanobacteriaN. flagelliformeis still unknown. In this study, we performed the first proteomic analysis of Kmal inN. flagelliformeunder different drought stresses using LC-MS/MS. In total, 421 malonylated lysine residues were found in 236 different proteins. GO and KEGG enrichment analysis indicated that these malonylated proteins were highly enriched in several metabolic pathways, including carbon metabolism and photosynthesis. Decreased malonylation levels were found to hinder the reception and transmission of light energy and CO2fixation, which led to a decrease in photosynthetic activity. Kmal was also shown to inhibit the flux of the TCA cycle and activate the gluconeogenesis pathway in response to drought stress. Furthermore, malonylated antioxidant enzymes and antioxidants were synergistically involved in reactive oxygen species (ROS) scavenging. Malonylation was involved in lipid degradation and amino acid biosynthesis as part of drought stress adaptation. This work represents the first comprehensive investigation of the role of malonylation in dehydratedN. flagelliforme, providing an important resource for understanding the drought tolerance mechanism of this organism.