BACKGROUND The aim of the study was to investigate whether levobupivacaine (LB) suppressed lipopolysaccharide (LPS)-induced high mobility group box 1 (HMGB1) release in vitro and in vivo, and to determin its molecular mechanisms of action. METHODS RAW264.7 cells were treated with LPS and LB for 24 h. Levels of HMGB1, nuclear factor-kappa B (NF-κB) and phosphorylated p38 mitogen-activated protein kinase (MAPK) were measured by Enzyme-linked immunosorbent assay and Western blotting; the levels of HMGB1 messenger RNA were measured by real-time polymerase chain reaction. In addition, cecal ligation and puncture-induced septic C57BL/6 received LB infusion, and the levels of HMGB1 and functional parameters of multiple organs determined using several detection kits. RESULTS LB inhibited HMGB1 release in vitro and in vivo. Furthermore, LB inhibited the translocation of NF-κB and phosphorylation of p38 MAPK in vitro. Mice treated with LB infusion improved survival in mice and significantly reduced cecal ligation and puncture-induced dysfunction of organs. CONCLUSIONS LB suppresses LPS-induced HMGB1 release in vitro and in vivo by partially inhibiting NF-κB/p38 MAPK pathways. LB can rescue mice from sepsis and protect against organ dysfunction in septic mice.