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Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease prevalent worldwide. This study investigated the effects of glycyrrhizin, an extract of licorice root, on the well-established model of 2,4-dinitrochlorobenzene-induced AD-like symptoms in mice. The severity of dermatitis, histopathological changes, serum IgE levels, changes in expression of high-mobility group box 1 (HMGB1), the receptor for advanced glycation end products (RAGE), nuclear factor (NF)-κB and inflammatory cytokines were evaluated. Treatment with glycyrrhizin inhibited the HMGB1 signaling cascade and ameliorated the symptoms of AD. Furthermore, in an in vitro study, the expression of RAGE was detected in a mouse mast cell line, P815 cells, and rmHMGB1 was found to be a potent inducer of mast cell activation by increasing Ca2+ influx, upregulating the CD117 and activating NF-κB signaling; these effects were also inhibited by glycyrrhizin. These findings implicate HMGB1 in the pathogenesis of AD and suggest that GL could be an effective therapeutic approach for cutaneous inflammation.HMGB1 expression was significantly increased in DNCB-induced mouse model of atopic dermatitis.HMGB1 was able to activate mast cells directly.Glycyrrhizin treatment ameliorated the symptoms of atopic dermatitis.