Endogenous ligands such as high‐mobility group box 1 (HMGB1) and nucleic acids are released by dying cells and bind Toll‐like receptors (TLRs). Because TLR9 sits at the interface of microbial and sterile inflammation by detecting both bacterial and endogenous DNA, we investigated its role in a model of segmental liver ischemia–reperfusion (I/R) injury. Mice were subjected to 1 hour of ischemia and 12 hours of reperfusion before assessment of liver injury, cytokines, and reactive oxygen species (ROS). Wild‐type (WT) mice treated with an inhibitory cytosine‐guanosine dinucleotide (iCpG) sequence and TLR9^−/− mice had markedly reduced serum alanine aminotransferase (ALT) and inflammatory cytokines after liver I/R. Liver damage was mediated by bone marrow–derived cells because WT mice transplanted with TLR9^−/− bone marrow were protected from hepatic I/R injury. Injury in WT mice partly depended on TLR9 signaling in neutrophils, which enhanced production of ROS, interleukin‐6 (IL‐6), and tumor necrosis factor (TNF). In vitro, DNA released from necrotic hepatocytes increased liver nonparenchymal cell (NPC) and neutrophil cytokine secretion through a TLR9‐dependent mechanism. Inhibition of both TLR9 and HMGB1 caused maximal inflammatory cytokine suppression in neutrophil cultures and conferred even greater protection from I/R injury in vivo. Conclusion: TLR9 serves as an endogenous sensor of tissue necrosis that exacerbates the innate immune response during liver I/R. Combined blockade of TLR9 and HMGB1 represents a clinically relevant, novel approach to limiting I/R injury. (HEPATOLOGY 2009.)