Nuclear DNA-binding protein high mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as sepsis and septic shock. Epi-sesamin (ESM), an important component of Asarum sieboldii roots, is known to exhibit anti-allergic, anti-nociceptive, and anti-fungal effects. However, little is known of its effects on HMGB1-mediated inflammatory responses. Here, we investigated this issue by monitoring the effects of ESM on lipopolysaccharide (LPS) or cecal ligation and the puncture (CLP)-mediated release of HMGB1, and on modulation of HMGB1-mediated inflammatory responses. ESM potently inhibited HMGB1 release, down-regulated HMGB1-dependent inflammatory responses in human endothelial cells, and inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with ESM resulted in reduced CLP-induced release of HMGB1 and sepsis-related mortality. Of particular interest, ESM inhibition of HMGB1-mediated anti-inflammatory activity was more potent than that by sesamin (SM), likely due to differences between their three-dimensional structures. These results indicate that ESM could be a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.