High mobility group box 1 (HMGB1) is an established inflammatory mediator when released from cells. Recent studies have implicated extracellular HMGB1 in the pathogenesis of various autoimmune diseases. The objective of this study was to determine whether HMGB1 could be a therapeutic target for experimental autoimmune encephalomyelitis (EAE). In this study, an anti-HMGB1 monoclonal antibody was injected intraperitoneally into a mouse model of EAE. We also measured serum cytokines levels in EAE and anti-HMGB1 monoclonal antibody-treated EAE. As a result, intraperitoneal injection of an anti-HMGB1 monoclonal antibody ameliorated the clinical and pathological severity of EAE and attenuated interleukin-17 up-regulation in serum. In conclusion, HMGB1 is involved in EAE pathogenesis and could trigger inflammation in the central nervous system. The novel aspect of this study is the demonstration that anti-HMGB1 ameliorates EAE. HMGB1 may be a novel therapeutic strategy for multiple sclerosis.