A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Histidine-rich glycoprotein (HRG) is a 75-kDa plasma protein which was recently proposed as a new biomarker to predict the outcome of sepsis patient and was demonstrated to improve the survival of septic mice through the regulation of neutrophils and vascular endothelial cells. Here, we monitored the effects of HRG on the lipopolysaccharide (LPS)-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in EA.hy 926 endothelial cells. Our results show that LPS induced significant HMGB1 translocation from nucleus to cytoplasma and large amount of HMGB1 release from EA. hy 926 endothelial cells which effectively inhibited by HRG. Furthermore, HRG potently inhibited high expression of adhesion molecules and release of inflammatory cytokines from HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with three pathogen-related pattern recognition receptors: TLR2 and TLR4 and RAGE. HRG also down-regulated the cell surface expression of all three HMGB1 receptors in endothelial cells. The protective effects of HRG in severe sepsis may partially be mediated through the inhibition of HMGB1 translocation/release.