BACKGROUND The release of high mobility group box 1 (HMGB1) induced by inflammatory signals acts as a cellular alarmin to trigger a chain of inflammatory responses. Although the inflammatory actions of HMGB1 are well studied, less is known about the therapeutic agents that can impede its release. This study investigated whether the isoflavonoid formononetin can modulate HMGB1 release in cellular inflammatory responses. METHODS RAW264.7 murine macrophages were exposed to lipopolysaccharide (LPS) in the presence or absence of formononetin. The levels of HMGB1 release, sirtuin 1 (SIRT1) expression, and HMGB1 acetylation were analyzed by immunoblotting and real-time polymerase chain reaction. The effects of resveratrol and sirtinol, an activator and inhibitor of SIRT1, respectively, on LPS-induced HMGB1 release were also evaluated. RESULTS Formononetin modulated cellular inflammatory responses by suppressing the release of HMGB1 by macrophages exposed to LPS. In RAW264.7 cells, formononetin significantly attenuated LPS-induced release of HMGB1 into the extracellular environment, which was accompanied by a reduction in its translocation from the nucleus to the cytoplasm. In addition, formononetin significantly induced mRNA and protein expression of SIRT1 in a peroxisome proliferator-activated receptor δ (PPARδ)-dependent manner. These effects of formononetin were dramatically attenuated in cells treated with small interfering RNA (siRNA) against PPARδ or with GSK0660, a specific inhibitor of PPARδ, indicating that PPARδ is involved in formononetin-mediated SIRT1 expression. In line with these effects, formononetin-mediated inhibition of HMGB1 release in LPS-treated cells was reversed by treatment with SIRT1-targeting siRNA or sirtinol, a SIRT1 inhibitor. By contrast, resveratrol, a SIRT1 activator, further potentiated the inhibitory effect of formononetin on LPS-induced HMGB1 release, revealing a possible mechanism by which formononetin regulates HMGB1 release through SIRT1. Furthermore, modulation of SIRT1 expression by transfection of SIRT1- or PPARδ-targeting siRNA significantly counteracted the inhibitory effects of formononetin on LPS-induced HMGB1 acetylation, which was responsible for HMGB1 release. CONCLUSIONS This study shows for the first time that formononetin inhibits HMGB1 release by decreasing HMGB1 acetylation via upregulating SIRT1 in a PPARδ-dependent manner. Formononetin consequently exhibits anti-inflammatory activity. Identification of agents, such as formononetin, which can block HMGB1 release, may help to treat inflammation-related disorders.