Rheumatoid arthritis (RA) is a systemic autoimmune disease that may cause bone damage and worsening disability. Manipulating antigen-specific Treg cells is a promising approach to treat autoimmune disease since the immune suppressive function of Treg cells has the feature of antigen specificity which could avoid overall immune suppression. It has been known that the function of Treg cells is impaired in RA, and adoptive transfer of Treg cells is effective in suppressing RA. Here, we designed a new approach to generate antigen-specific Treg cells by culturing CD4 T cells from mice with RA onset, and we also proved that the adoptive transfer of these antigen-specific Treg cells reversed the collagen-induced arthritis (CIA) progression by suppressing the key inflammatory cytokine TNF-α. Further analysis showed that the transferred Treg cells were stable in vivo. These findings suggest this novel approach may have clinical applications for treatment of autoimmunity, including RA and other autoimmune disorders.