AbstractTriptolide, a diterpene triepoxide, is one of the major components of most functional extracts of Tripterygium wilfordii Hook f, which is known to have various biological effects, including immunosuppressive, anti‐inflammatory and anti‐tumor functions. We studied the inhibitory effect of triptolide on endotoxemia (ETM)‐induced oxidative stress, which was induced in C57BL/6 mice by lipopolysaccharide (LPS) and D‐galactosamine (D‐GalN). Pretreatment with triptolide decreased the reactive oxygen species (ROS) levels, mortality rate and liver injury after LPS/D‐GalN injection. We utilized comprehensive proteomics to identify alterations in liver protein expression during pretreatment with triptolide or N‐acetylcysteine (NAC) after LPS/D‐GalN injection, 44 proteins were found to be related to oxidative stress, mitochondria, metabolism and signal transduction, and 23 proteins of them seemed to be significantly up‐ or down‐regulated. Furthermore, both triptolide and NAC inhibited activation of c‐jun NH2‐terminal kinases (JNK) and mitogen‐activated protein kinase p38 (p38), phosphorylation of inhibitor of nuclear factor‐kappa B (IκB) and activation of nuclear factor‐κB (NF‐κB). These results demonstrated that triptolide inhibited the activation of JNK and p38 by decreasing ROS levels, which in turn inhibited the hepatic injury. In addition, we set and validated the phosphorylation model of extracellular signal‐regulated kinase (ERK) and proposed that triptolide probably induced ERK phosphorylation through inhibiting its dephosphorylation rates. These results showed that triptolide can effectively reduce the oxidative stress and partially rescue the damage in the liver induced by LPS/D‐GalN. J. Cell. Biochem. 113: 1022–1033, 2012. © 2011 Wiley Periodicals, Inc.