Signals from stressed cells and the enteric microbiota activate macrophages and dendritic cells and mediate intestinal inflammation. HMGB1 serves as an immunogenic stimuli causing release of inflammatory cytokines by myeloid cells. Ethyl pyruvate inhibits secretion of HMGB1 and improves survival in models of endotoxemia and hemorrhagic shock. We reasoned that ethyl pyruvate may be protective in colitis, which involves similar inflammatory pathways. In IL-10(-/-) mice with established chronic colitis, ethyl pyruvate administration ameliorated colitis and reduced intestinal cytokine production. IL-10(-/-) mice demonstrated increased intestinal HMGB1 expression and decreased expression of RAGE compared with wild-type mice. Fecal HMGB1 levels were decreased in ethyl pyruvate-treated mice. Furthermore, ethyl pyruvate induced HO-1 expression in intestinal tissue. In TNBS-induced colitis, intrarectal administration of ethyl pyruvate resulted in amelioration of colitis and reduced intestinal cytokine production. In LPS-activated murine macrophages, ethyl pyruvate decreased expression of IL-12 p40 and NO production but did not affect IL-10 levels. Ethyl pyruvate did not inhibit nuclear translocation of NF-kappaB family members but attenuated NF-kappaB DNA binding. Additionally, ethyl pyruvate induced HO-1 mRNA and protein expression and HO-1 promoter activation. Moreover, ethyl pyruvate prevented nuclear-to-cytoplasmic translocation of HMGB1. In conclusion, the HMGB1/RAGE pathway has pathophysiologic and diagnostic significance in experimental colitis. Ethyl pyruvate and other strategies to inhibit HMGB1 release and function represent promising interventions in chronic inflammatory diseases.