The extracellular matrix of blood vessels is well-known to contribute to multiple facets of vascular homeostasis and disease (Xu, Shi, Biochim Biophys Acta 1842:2106–2119, 2014; Lin, Davis, Arterioscler Thromb Vasc Biol 43:1599–1616, 2023). Over the past three decades, however, there has been increasing recognition that the vascular matrix is not limited to the medial and adventitial spaces of blood vessels. Indeed, the endovascular surface of the vascular intima also features a luminal matrix of substantial importance to health and disease: the endothelial glycocalyx. In this review, we will discuss what is known about the structure of the endothelial glycocalyx and its substantial heterogeneity as well as the key physiological functions of this endothelial surface layer. We will also review how the endothelial glycocalyx is damaged during acute and chronic vascular diseases, with a focus on what has been learned from human studies, including an increasing literature of randomized controlled studies testing the efficacy of glycocalyx-protective therapeutics.