High-mobility group box 1 (HMGB1) was recently established as a proinflammatory mediator of sepsis, and its potential role in the pathogenesis of sepsis remains elusive. In the present study, we determined whether HMGB1 increases the permeability of the endothelial cell monolayer in sepsis. Permeability was measured from fluorescein isothiocyanate (FITC)-dextran 40-kDa flux across the endothelial cell monolayer at control and after HMGB1 administration. We found that HMGB1 increased human umbilical vein endothelial cell permeability to FITC-dextran 40 kDa in a time- and concentration-dependent manner. HMGB1 induced the mRNA transcription and protein expression of receptor for advanced glycation end products (RAGE). Blockade of cell surface receptors RAGE with specific neutralizing antibodies and RAGE siRNA or blockade of Src family tyrosine kinase with inhibitor PP2 significantly reduced HMGB1-induced hyperpermeability of endothelial cell monolayer. Our data demonstrate that (1) HMGB1 increases permeability of endothelial cell monolayer in a time- and concentration-dependent manner and (2) HMGB1-induced hyperpermeability is mediated through RAGE and Src family tyrosine kinase signaling pathway. These findings may have implications for therapeutic interventions in patients with sepsis.