Patients with diabetes mellitus are characterized by an extraordinary vascular vulnerability. Traditionally, glucose-induced damage to the vascular endothelium is believed to be one of the first steps in the development of vascular damage. However, in the healthy vessel the endothelium is protected by a matrix layer of highly glycosylated proteins that form a physical barrier between the endothelium and the blood flowing past. Although its presence has been known for half a century, this so-called glycocalyx earned little attention from researchers in the past because of an underestimation of its size. In the last decade it has become clear that its full thickness actually exceeds that of the vascular endothelium. Accumulating research into the functional relevance of the endothelial glycocalyx suggests an important role for this layer in the development of cardiovascular disease in diabetes mellitus. Here we will present an overview of the biochemistry of the intact glycocalyx, current methods to assess the glycocalyx, and its possible role in the pathophysiology of cardiovascular disease in diabetes.