Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.