There is increasing evidence that endoplasmic reticulum (ER) stress contributes to the development of atherosclerosis in diabetes mellitus. The purpose of this study was to determine the effects of increased hexosamine biosynthesis pathway (HBP) flux on ER stress levels and the complications of ER stress associated with diabetes and atherosclerosis in hepatic cells. Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, was overexpressed in HepG2 cells by use of an adenoviral expression system. The ER stress response and downstream effects, including activation of lipid and inflammatory pathways, were determined using real-time PCR, immunoblot analysis, and cell staining techniques. GFAT overexpression resulted in increased expression of ER stress markers, including Grp78, Grp94, calreticulin, and GADD153, relative to cells infected with an empty adenoviral vector. In addition, GFAT overexpression promoted lipid, but not cholesterol, accumulation in hepatic cells as well as inflammatory pathway activation. Treatment with 6-diazo-5-oxo-norleucine, a GFAT antagonist, blocked the effects of GFAT overexpression. Consistent with our in vitro data, hyperglycemic mice presented with elevated markers of hepatic ER stress, glucosamine and lipid accumulation. Together, these data suggest that HBP flux-induced ER stress plays a role in the development of hepatic steatosis and atherosclerosis under conditions of hyperglycemia.