“Metabolic memory” is identified as a phenomenon that transient hyperglycemia can be remembered by vasculature for quite a long term even after reestablishment of normoglycemia. NADPH oxidases (Noxs) and endothelial nitric oxide synthase (eNOS) are important enzymatic sources of reactive oxygen species (ROS) in diabetic vasculature. The aim of this study is to explore the roles of epigenetics and ROS derived from Noxs and eNOS in the metabolic memory. In this study, we demonstrated that vascular ROS was continuously activated in endothelium induced by transient high glucose, as well as sustained vascular endothelial dysfunction. The Nox4 and uncoupled eNOS are the major sources of ROS, while inhibition of Nox4 and eNOS significantly attenuated oxidative stress and almost recovered the endothelial function in metabolic memory. Furthermore, the aberrant histone methylation (H3K4me1, H3K9me2, and H3K9me3) at promoters of Nox4 and eNOS are the main causes for the persistent up-regulation of these two genes. Modifying the histone methylation could reduce the expression levels of Nox4 and eNOS, thus obviously attenuating endothelial dysfunction. These results indicate that histone methylation of Nox4 and eNOS play a key role in metabolic memory and may be the potential intervention targets for metabolic memory.