Oncometabolites including D-2-hydroxyglutarate (D2HG) and L2HG have come under the spotlight due to their roles in promoting tumorigenesis. These oncometabolites dysregulate growth-signalling pathways by noncovalently interacting with key regulators. However, it is unclear whether D/L 2HG influence cellular functions through covalent protein modification. Here we discovered a novel type of posttranslational modification by a novel unbiased immobilized metal ion affinity chromatographic (IMAC) based strategy and further confirmed it as the O-2-Hydroxyglutarylation. We found D2HG modification undergo upregulation upon D2HG treatment or in IDH mutant cells, while L2HG modification upregulate upon L2HG treatment or in hypoxia environment. By differential proteomics, we distinguished the enantiomer D2HG and L2HG modifications and constructed D2HG and L2HG modification datasets, and some of them were validated by isotopic metabolic flux approach. Quantitative proteomics identified O-2-Hydroxyglutarylation sites in multiple functional proteins, including DNA replicators and responders to oxidative stress. Two kinase, MRCKA bearing D2HG modification at site S794 and SLK bearing L2HG at S719 have been identified and confirmed by synthetic peptide standards. Phosphoproteomics revealed that the phosphorylation level of MRCKA and SLK's substrate downregulated in D2HG and L2HG accumulated cells respectively, suggesting a inhibitory effect of 2HG modification on kinase. The discovery of O-2-Hydroxyglutarylation shed light on the intricate relationship between cancer progression and the oncometabolites D/L-2HG, opening new pathways for the development of more effective cancer treatments.