High-mobility group box 1 (HMGB1) was recently shown to be an important extracellular mediator of systemic inflammation, and endothelial cell protein C receptor (EPCR) has been shown to be involved in vascular inflammation. Hyperoside is an active compound isolated from Rhododendron brachycarpum G. Don (Ericaceae) that was reported to have anti-oxidant, anti-hyperglycemic, anti-cancer, and anti-coagulant activities. Here, we show, for the first time, the anti-septic effects of hyperoside in HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo. The data showed that hyperoside posttreatment suppressed lipopolysaccharide (LPS)-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangement. Hyperoside also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice and phorbol-12-myristate 13-acetate (PMA) of cecal ligation and puncture (CLP)-induced EPCR shedding. In addition, hyperoside inhibited the production of tumor necrosis factor-α (TNF-α) and the HMGB1-mediated activation of Akt, nuclear factor-κB (NF-κB), and extracellular regulated kinase (ERK) 1/2 in HUVECs. Hyperoside also reduced the CLP-induced release of HMGB1, the production of interleukin (IL)-1β, and septic mortality. Collectively, these results suggest hyperoside as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.