Itaconate is an important anti-microbial and immunoregulatory metabolite involved in host-pathogen interactions. A key mechanistic action of itaconate is through the covalent modification of cysteine residues via Michael addition, resulting in “itaconation”. However, it is unclear whether itaconate has other regulatory mechanisms. In this work, we discovered a novel type of post-translational modification by promiscuous antibody enrichment and data analysis with the open-search strategy, and further confirmed it as the lysine “itaconylation”. We showed that itaconylation undergo significant up-regulation upon lipopolysaccharides (LPS) stimulation in RAW264.7 macrophages. Quantitative proteomics identified itaconylation sites in several functional proteins, including glycolytic enzymes and histones, some of which were confirmed by synthetic peptide standards.