Glucosamine is an essential substrate for N-linked protein glycosylation. However, elevated levels of glucosamine can induce endoplasmic reticulum (ER) stress. Glucosamine-induced ER stress has been implicated in the development of diabetic complications, including atherosclerosis and hepatic steatosis. In this study, we investigate the potential relationship between the effects of glucosamine on lipid-linked oligosaccharide (LLO) biosynthesis, N-linked glycosylation, and ER homeostasis. Mouse embryonic fibroblasts (MEFs) were cultured in the presence of 0–5 mM glucosamine for up to 18 h, and LLO biosynthesis was monitored by fluorescence-assisted carbohydrate electrophoresis. ER stress was determined by quantification of unfolded protein response (UPR) gene expression. We found that exposure of MEFs to ≥1 mM glucosamine significantly impaired the biosynthesis of mature (Glc3Man9GlcNAc2) LLOs before the activation of the UPR, which resulted in the accumulation of an LLO intermediate (Man3GlcNAc2). The addition of 4-phenylbutyric acid (4-PBA), a chemical chaperone, was able to alleviate ER stress but did not rescue LLO biosynthesis. Other ER stress-inducing agents, including dithiothreitol and thapsigargin, had no effect on LLO levels. Together, these data suggest that elevated concentrations of glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. We hypothesize that this pathway represents a causative link between hyperglycemia and the development of diabetic complications.