High mobility group box protein 1 (HMGB1) modulates the innate immune response when present in the extracellular compartment. Receptors for HMGB1 include TLR4, TLR2, and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce LPS tolerance. HMGB1 dose-response experiments were performed on IFN-γ-differentiated human monocyte-like THP-1 cells. Treatment with 1 μg/ml HMGB1 18 h before exposure to LPS (1 μg/ml) decreased TNF release, NF-κB nuclear DNA-binding activity, phosphorylation, and degradation of IκBα. Preconditioning with HMGB1 alone and HMGB1 in the presence of polymyxin B decreased LPS-mediated, NF-κB-dependent luciferase reporter gene expression. The specificity of HMGB1 in tolerance induction was supported further by showing that boiled HMGB1 failed to induce tolerance, and antibodies against HMGB1 blocked the induction of LPS tolerance. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type mice became LPS-tolerant following HMGB1 exposure ex vivo, but macrophages derived from RAGE-deficient mice failed to develop tolerance and responded normally to LPS. Mice preconditioned with HMGB1 (20 μg) 1 h before LPS injection (10 mg/kg) had lower circulating TNF compared with control mice preconditioned with saline vehicle. Similarly, decreased nuclear DNA binding of hepatic NF-κB was observed in mice preconditioned with HMGB1. Taken together, these results suggest that extracellular HMGB1 induces LPS tolerance, and the RAGE receptor is required for this induction.