Severe sepsis is a life-threatening complication of infection and injury affecting more than 700,000 people in the United States each year. Two thirds of patients with severe sepsis will survive to be discharged. Survivors have high incidence of cognitive impairment, immune dysregulation, functional impairments with marked disability, and 5-year mortality rates of 82%. High-mobility group box 1 (HMGB1) is necessary and sufficient mediator of sepsis pathogenesis in experimental models of this syndrome. The spleen is a crucial organ in the immune response to severe infection, and splenocyte dysfunction occurs in sepsis survivors. We hypothesized that HMGB1 plays a key role in mediating the immune dysfunction of splenocytes in sepsis survivors. Mice that survived cecal ligation and puncture-induced sepsis develop persistent splenomegaly; furthermore, splenocytes derived from sepsis survivors had enhanced responses to lipopolysaccharide ex vivo. Administration of neutralizing anti-HMGB1 antibody to sepsis survivors attenuated development of splenomegaly and reversed splenocyte priming. Splenocytes exposed to HMGB1 and subsequently challenged with cognate ligands to Toll-like receptor 2 (TLR2,) TLR4, TLR9, and RAGE (receptor for advanced glycation end product) receptors had enhanced cytokine release as compared with splenocytes not previously exposed to HMGB1. Exposure of TLR2, TLR9, or RAGE splenocytes to HMGB1 enhanced responses to other TLR receptor ligands; in contrast, HMGB1 failed to prime TLR4 splenocytes. These findings indicate that exposure to HMGB1 enhances splenocyte responses to secondary inflammatory challenges, a priming effect dependent on TLR4, and that anti-HMGB1 monoclonal antibody may be beneficial in sepsis survivors.