Damage to the endothelial glycocalyx, which helps maintain vascular homeostasis, heightens the sensitivity of the vasculature to atherogenic stimuli. Patients with renal failure have endothelial dysfunction and increased risk for cardiovascular morbidity and mortality, but the state of the endothelial glycocalyx in these patients is unknown. Here, we used Sidestream Darkfield imaging to detect changes in glycocalyx dimension in dialysis patients and healthy controls from in vivo recordings of the sublingual microcirculation. Dialysis patients had increased perfused boundary region and perfused diameters, consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties. These patients also had higher serum levels of the glycocalyx constituents hyaluronan and syndecan-1 and increased hyaluronidase activity, suggesting the shedding of these components. Loss of residual renal function had no influence on the imaging parameters but did associate with greater shedding of hyaluronan in blood. Furthermore, patients with higher levels of inflammation had more significant damage to the glycocalyx barrier. In conclusion, these data suggest that dialysis patients have an impaired glycocalyx barrier and shed its constituents into blood, likely contributing to the sustained endothelial cell activation observed in ESRD.