We have previously shown that glucosamine promotes endoplasmic reticulum (ER) stress in vascular cells leading to both inflammation and lipid accumulation--the hallmark features of atherosclerosis. Pretreatment with glycogen synthase kinase (GSK)-3 inhibitors protects cultured cells from ER stress-induced dysfunction. Here we evaluate the potential role of GSK-3 on the pro-atherogenic effects of hyperglycemia and ER stress. We show that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol under conditions of ER stress. Furthermore, GSK-3 inhibitors, including valproate, attenuate ER stress-induced unesterified cholesterol accumulation in wild-type mouse embryonic fibroblasts. In vivo we show that hyperglycemic apoE-deficient mice have accelerated atherogenesis at the aortic root compared with normoglycemic control mice. Mice fed a diet supplemented with 625 mg/kg valproate have significantly reduced lesion volume relative to nonsupplemented controls. Valproate supplementation has no apparent effect on the plasma levels of either glucose or lipids or on the expression of diagnostic markers of ER stress in the lesion. Significant reductions were observed in total hepatic lipids (>50.4%) and hepatic GSK-3beta activity (>55.8%) in mice fed the valproate diet. In conclusion, dietary supplementation with low levels of valproate significantly attenuates atherogenesis in hyperglycemic apoE-deficient mice. The in vivo anti-atherogenic effects of valproate are consistent with its ability to inhibit GSK-3 and interfere with pro-atherogenic ER stress signaling pathways in vitro.