Diabetes is a pivotal risk factor for cardiovascular disease as well as microvascular complications, including retinopathy and nephropathy. Chronic hyperglycemia is a key player in linking diabetes with endothelial dysfunction which, in turn, contributes to cardiovascular disease. Indeed, hyperglycemia acts as a trigger for endothelial dysfunction, promoting a shift in the endothelium from a protective, anti-inflammatory state to a dysfunctional, injury-prone phenotype. A hyperglycemic environment triggers several pathogenetic mechanisms, including alterations in bioenergetics, production of advanced glycation end products, oxidative stress and mitochondrial dysfunction, all contributing to endothelial dysfunction. The activation of these pathophysiological mechanisms by hyperglycemia culminates in reduced nitric oxide production, as well as the induction of oxidative stress and inflammation, all of which are pivotal in impairing endothelial homeostasis and promoting cellular damage. Besides these classical mechanisms, there is growing attention on novel pathogenetic factors linking diabetic hyperglycemia with endothelial dysfunction, such as the ACE2 protein. The latter is emergeing for its potential to counter hyperglycemia-induced cellular damage through its vasoprotective and anti-inflammatory actions, making it a promising therapeutic target for tackling endothelial dysfunction. This review provides an overview of classical as well as emerging mechanisms underpinning the deleterious effects of diabetic hyperglycemia on endothelial dysfunction. In turn, understanding the molecular interconnections between hyperglycemia and endothelial dysfunction is crucial for developing novel strategies to restore endothelial homeostasis and mitigate diabetic vascular complications.