Updated project metadata. Lysine malonylation (Kmal) is a new posttranslational modification (PTM), which has been reported in several prokaryotic and eukaryotic species. Although Kmal can regulate many and diverse biological processes in various organisms, knowledge about this important PTM in the apicomplexan parasite Toxoplasma gondii is limited. In this study, we performed the first global profiling of malonylated proteins in T. gondii tachyzoites using affinity enrichment and LC-MS/MS analysis. Three experiments performed in tandem revealed 331, 367, 373 Kmal sites on 227, 252, 241 malonylated proteins, respectively. Computational analysis showed the identified malonylated proteins to be localized in various subcellular compartments and involved in many cellular functions, particularly mitochondrial function and oxidation of fatty acids. Additionally, two conserved Kmal motifs with a strong bias for cysteine were detected. Taken together, these findings provide the first report of Kmal profile in T. gondii and should be an important resource for studying the physiological roles of Kmal in this parasite.