Foxp3+ regulatory T (Treg) cells, as one of the subtypes of CD4+ T cells, are the crucial gatekeeper in the pathogenesis of self-antigen reactive diseases. In this context, we demonstrated that the selective ablation of early growth response gene 1 (Egr-1) in CD4+ T cells exacerbated experimental autoimmune encephalomyelitis (EAE) in murine models. The absence of Egr-1 in CD4+ T cells, obtained from EAE mice and naïve CD4+ T cells, impeded the differentiation and influence of Treg. Importantly, in CD4+ T cells of multiple sclerosis patients, both Egr-1 and Foxp3 were found to decrease. Further studies showed that distinct from the classical Smad3 route, TGF-β could activate Egr-1 through the Raf-Erk signaling route to promote Foxp3 genetic modulation, thereby promoting Treg cell differentiation and reducing EAE inflammation. A novel natural Egr-1 agonist, calycosin, was found to attenuate EAE progression by regulating the differentiation of Treg. Together, the above results indicate the value of Egr-1, as a novel Foxp3 transactivator, for the differentiation of Treg cells in the development of self-antigen reactive diseases.