Primary immune thrombocytopenia (ITP) is the most common acquired autoimmune bleeding disorder. Abnormally increased levels of High Mobility Group Box 1 (HMGB1) protein associate with thrombocytopenia and therapeutic outcome in ITP. Previous studies proposed that a natural inhibitor of HMGB1, 18β-glycyrrhetinic acid (18β-GA), could be used for its anti-inflammatory and immune-modulatory effects, although its ability to correct immune balance in ITP is unclear. In this study, we showed that plasma HMGB1 correlated negatively with platelet counts in ITP patients, and confirmed that 18β-GA stimulated the production of regulatory T cells (Treg), restored the balance of CD4+ T-cell subsets and enhanced the suppressive function of Treg through blocking the effect on HMGB1 in patients with ITP. HMGB1 short hairpin RNA interference masked the effect of 18β-GA in Treg of ITP patients. Furthermore, we found that 18β-GA alleviated thrombocytopenia in mice with ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The proportion of circulating Treg increased significantly, while the level of plasma HMGB1 and serum antiplatelet antibodies decreased significantly in ITP mice along 18β-GA treatment. In addition, 18β-GA reduced phagocytic activity of macrophages towards platelets both in ITP patients and ITP mice. These results indicate that 18β-GA has the potential to restore immune balance in ITP via inhibition of HMGB1 signaling. In short, this study reveals the role of HMGB1 in ITP, which may serve as a potential target for thrombocytopenia therapy.