The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long‐chain acyl‐CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH‐7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK‐3β inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH‐7 cells. shRNA that target GSK‐3β also inhibited the upregulation of ACSL3 and lipid accumulation in HuH‐7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK‐3β inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK‐3β represent novel therapeutic targets for lipid dysregulation by ER stress. J. Cell. Biochem. 112: 881–893, 2011. © 2010 Wiley‐Liss, Inc.