© 2020 Elsevier Ltd The post-translational modification of serine and threonine residues of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates diverse cellular processes in the cardiovascular system. UDP-GlcNAc is a substrate for O-GlcNAc transferase, which catalyzes the attachment of O-GlcNAc to proteins. O-GlcNAcase catalyzes the removal of O-GlcNAc from proteins. UDP-GlcNAc is the end product of the hexosamine biosynthesis pathway, which is regulated primarily by glucose-6-phosphate-Glutamine:fructose-6-phosphate amidotransferase (GFAT). GFAT catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. Whereas O-GlcNAc is essential for cell viability, sustained increases in O-GlcNAc levels have been implicated in the etiology of many chronic diseases and is associated with glucose toxicity and diabetic complications in various organs including the cardiovascular system. This review provides an overview of the regulation of protein O-GlcNAcylation followed by a discussion of potential mechanisms by which dysregulation in O-GlcNAc cycling contributes to the adverse effects of diabetes on the cardiovascular system.