High mobility group box 1 (HMGB1) protein is a crucial cytokine that mediates response to infection, injury, and inflammation. Rosmarinic acid (RA) is an important component of the leaves of Perilla frutescens and has neuroprotective, anti-microbial, anti-oxidant, and anti-cancer effects but little is known of its effects on HMGB1-mediated inflammatory response. Here, we investigated this issue by monitoring the effects of RA on the lipopolysaccharide (LPS) or cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated modulation of inflammatory responses. RA potently inhibited the release of HMGB1 and down-regulated HMGB1-dependent inflammatory responses in human endothelial cells. RA also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. Furthermore, RA reduced CLP-induced HMGB1 release and sepsis-related mortality. Given these results, RA should be viewed as a candidate therapeutic agent for the treatment of various inflammatory diseases via inhibition of the HMGB1 signaling pathway.