Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidence by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 as compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1alpha. Intraperitoneal injection of damaged-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared to Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damaged-associated molecular patterns. However, upon stimulation with damaged-associated molecular patterns the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts, an effect accompanied by weaker release of several cytokines, including IL-1alpha, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.