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Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4+CD25+Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4+CD25+Foxp3+Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4+CD25+Foxp3+Treg cells in the thymus upon indirubin treatment. Furthermore, CD4+CD25+Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4+CD25+Foxp3+Treg cell level with preserving immunosuppressive function.