Abstract Introduction Posttranslational modifications of histones play a critical role in regulating gene expression in health and disease. Activation of histone methyltransferase enhancer of zeste homolog 2 (EZH2) catalyses repressive trimethylation of histone 3 at lysine residue 27 (H3K27me3) and favours reactive oxygen species (ROS) generation as well as pro-inflammatory changes. Oxidative stress and inflammation are linked to hyperglycaemia-induced endothelial dysfunction. Purpose The present study was designed to investigate whether EZH2 may represent a novel therapeutic target against hyperglycaemia-induced abnormal phenotypes in the endothelium. Methods The effect of EZH2 was tested in aortas from male mice (3–5 months old), human aortic endothelial cells (HAECs) and aortic endothelial cells isolated from diabetic individuals (D-HAECs) in the presence and in the absence of pharmacological inhibitor GSK126 (5 μM) or gene silencing of EZH2. HAECs and aortic rings were exposed either to normal glucose (5 mM) or high glucose (25 mM) for 20 hrs. Expression levels of H3K27me3, EZH2, oxidant and pro-inflammatory genes were determined by RT-PCR, western blotting, and protein activation assay kits. Superoxide anion production was measured by electron spin resonance spectroscopy. Binding of H3K27me3 to the promoter region of candidate genes was evaluated by chromatin immunoprecipitation (ChIP). Endothelium-dependent and independent relaxations were assessed in aortic rings by organ chamber experiments. Results Immunoblotting showed a significant increase in EZH2 activity measured as H3K27me3 in HAECs exposed to high glucose and in D-HAEC. ChIP assays showed that binding of H3K27me3 to the promoter of anti-oxidant and transcription factor JunD genes triggers ROS generation and activation of inflammatory pathways. Interestingly enough, histone methyltransferase EZH2 inhibitor GSK126 or specific siRNA blunting H3K27me3 abolished hyperglycaemia-induced oxidative stress and inflammation. Moreover, targeting EZH2 rescued endothelial function. Conclusions These results uncover adverse epigenetic signatures underlying endothelial dysfunction in diabetes. Targeting EZH2 may attenuate oxidative and inflammatory transcriptional programmes and thus prevent vascular disease in this setting. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Research Council, the Swedish Heart & Lung Foundation, and King Gustav V and Queen Victoria Foundation.