Sepsis is a state of disrupted inflammatory homeostasis that is initiated by infection. High mobility group box 1 (HMGB1) protein acting as a late mediator of severe vascular inflammatory conditions, such as sepsis and endothelial cell protein C receptor (EPCR), is involved in vascular inflammation. Fisetin, an active compound from the family Fabaceae, was reported to have antiviral, neuroprotective, and anti-inflammatory activities. Here, we determined the anti-septic effects of fisetin on HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo, for the first time. First, we monitored the effects of post-treatment fisetin on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment fisetin was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. Fisetin also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. Fisetin induced potent inhibition of phorbol-12-myristate 13-acetate (PMA) and CLP-induced EPCR. Fisetin also inhibited the expression and activity of tumor necrosis factor-α converting enzyme, induced by PMA in endothelial cells. In addition, fisetin inhibited the production of tumor necrosis factor-α and the activation of AKT, nuclear factor-κB, and extracellular regulated kinases 1/2 by HMGB1 in HUVECs. Fisetin also down-regulated CLP-induced release of HMGB1, production of interleukin 1β, and reduced septic mortality. Collectively, these results suggest that fisetin may be a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.