p-Cresyl sulfate (PCS) is a risk factor of cardiovascular disease in patients with chronic kidney disease. Here we tested whether serum PCS levels were related to the rate and evolution of carotid atherosclerosis in hemodialysis patients and identified a potential mechanism. A total of 200 hemodialysis patients were categorized as with or without carotid atherosclerotic plaque and followed for 5 years. Serum PCS levels were found to be higher in patients with than without carotid atherosclerotic plaque and positively correlated with increased total plaque area during follow-up. Multiple logistic regression and mixed effects model analyses showed that serum PCS levels were independently associated with the incidence and progression of carotid atherosclerotic plaque. PCS induced inflammatory factor and adhesion molecule expression in endothelial cells and macrophages. In addition, PCS triggered monocyte-endothelial cell interaction in vitro and in vivo through increased production of reactive oxygen species. Compared with controls, increase of PCS levels produced by gavage promoted atherogenesis in 5/6-nephrectomized apoE-/- mice; a process attenuated by NADPH oxidase inhibitors. Thus, increased serum PCS levels are associated with the occurrence and progression of carotid atherosclerosis in hemodialysis patients and promote atherogenesis through increased reactive oxygen species production.