Dysregulation of the immune system and impairment in the function and number of patient-derived regulatory T cells (Treg) have an important role in multiple sclerosis (MS) pathogenesis. MS patients still receive different medications to overcome the relapses and to slow the disease progression. However, the benefits of these therapies are limited and are accompanied by different side effects. The immunoregulatory effects of Silymarin as a plant-derived flavonoid have shown in studies. In the present study, regulatory T cells (Tregs) were isolated from MS patients who diagnosed as new cases and IFN-β-treated RRMS patients. Isolated Treg cells were cultured in the presence of different concentrations of Silymarin (50, 100, 150μM) for 48, 72, and 120h. Proliferation and activation of Treg cells were assessed by flow cytometry. Also, FOXP3, JAK3, and STAT5 gene expression, IL-10, and TGF-β production by Tregs were evaluated by real-time PCR and ELISA respectively. The results showed that Silymarin promoted Treg proliferation at 100μM concentration after 72h. Additionally, IL-10, TGF-β levels, and FOXP3, JAK3, and STAT5 gene expression enhanced by Silymarin dose and time dependently. Our preliminary results suggest that the induction and activation of Tregs could be an underlying mechanism of the ancient used herbal medicine Silymarin, providing effective means against autoimmune and inflammatory diseases.