Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure in the United States and is primarily mediated by toxic metabolites that accumulate in the liver upon depletion of glutathione stores. However, cells of the innate immune system, including natural killer (NK) cells, neutrophils, and Kupffer cells, have also been implicated in the centrilobular liver necrosis associated with APAP. We have recently shown that dendritic cells (DCs) regulate intrahepatic inflammation in chronic liver disease and, therefore, postulated that DC may also modulate the hepatotoxic effects of APAP. We found that DC immune‐phenotype was markedly altered after APAP challenge. In particular, liver DC expressed higher MHC II, costimulatory molecules, and Toll‐like receptors, and produced higher interleukin (IL)‐6, macrophage chemoattractant protein‐1 (MCP‐1), and tumor necrosis factor alpha (TNF‐α). Conversely, spleen DC were unaltered. However, APAP‐induced centrilobular necrosis, and its associated mortality, was markedly exacerbated upon DC depletion. Conversely, endogenous DC expansion using FMS‐like tyrosine kinase 3 ligand (Flt3L) protected mice from APAP injury. Our mechanistic studies showed that APAP liver DC had the particular capacity to prevent NK cell activation and induced neutrophil apoptosis. Nevertheless, the exacerbated hepatic injury in DC‐depleted mice challenged with APAP was independent of NK cells and neutrophils or numerous immune modulatory cytokines and chemokines. Conclusion: Taken together, these data indicate that liver DC protect against APAP toxicity, whereas their depletion is associated with exacerbated hepatotoxicity. (HEPATOLOGY 2011;)