Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD (P) H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD (P) H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD (P) H oxidase activation, in the form of phosphorylation of p47 phox subunit of NAD (P) H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47 phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD (P) H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD (P) H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.