Endoplasmic reticulum stress (ER stress) has been increasingly recognized as an important mechanism in various liver diseases. However, its intrinsic physiological role in acute liver failure (ALF) remains largely undetermined. This study aimed to examine how ER stress orchestrates glycogen synthase kinase 3β (GSK3β) and inflammation to affect ALF. In a murine ALF model induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS), 4-phenylbutyric acid (4-PBA) is to be administered to relieve ER stress. The lethality rate, liver damage, cytokine expression, and the activity of GSK3β were evaluated. How to regulate LPS-induced inflammation and TNF-α-induced hepatocyte apoptosis by ER stress was investigated in vitro. In vivo, ER stress was triggered in the liver with the progression of mice ALF model. ER stress was essential for the development of ALF because ER stress inhibition by 4-PBA ameliorated the liver damage through decreasing liver inflammation and hepatocyte apoptosis. 4-PBA also decreased GSK3β activity in the livers of ALF mice. In vitro, ER stress induced by tunicamycin synergistically increased LPS-triggered pro-inflammatory cytokine induction and promoted the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway in bone marrow-derived macrophages; moreover, tunicamycin also cooperated with TNF-α to increase hepatocyte apoptosis. ER stress promoted LPS-triggered inflammation depending on GSK3β activation because inhibition of GSK3β by SB216763, the specific inhibitor of GSK3β, resulted in downregulation of pro-inflammatory genes. ER stress contributes to liver inflammation and hepatotoxicity in ALF, particularly by regulating GSK3β, and is therefore a potential therapeutic target for ALF.