Abstract : Acetaminophen-induced drug-induced acute liver injury is a liver disease which is one of the most serious human health diseases. Dehydroandrographolide is a natural product found in Andrographis paniculata (Burm.f.) Wall., Acanthaceae, with therapeutical uses such as analgesic and hepatoprotective. The present study was designed to analyze the effect of dehydroandrographolide on acetaminophen-induced acute liver failure. The results showed that dehydroandrographolide attenuated the damage caused by acetaminophen-induced acute liver failure in C57BL/6 mice through a decreased level of serum aspartate aminotransferase and glutamic-pyruvic transaminase. In addition, dehydroandrographolide increased the levels of glutathione and significantly increased serum superoxide dismutase. In vitro, dehydroandrographolide significantly activating nuclear-related factor 2, glutamate-cysteine ligase, glutamate-cysteine ligase modifier, heme oxygenase-1, and NAD(P)H quinone dehydrogenase 1 proteins promoted the phosphorylation of acetyl-CoA carboxylase, and 5′ adenosine monophosphate–activated protein kinase (α and β), inhibited the phosphorylation of phosphoinositide 3-kinase, serine/threonine kinase, and mammalian target of rapamycin. These results suggest that dehydroandrographolide can improve acetaminophen-induced drug-induced acute liver failure in mice.Graphical abstract: