T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4+ T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4+ CD25+ regulatory T cells (T reg) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T reg. In this study, we demonstrate that after a primary Ag challenge, T reg proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T reg after primary Ag challenge augmented the total suppressive function of T reg against TCR repertoire modification but not against the proliferation of memory CD4+ T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T reg, which would be crucial for limiting excess primary and memory CD4+ T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T reg is an ideal strategy for the clinical use of T reg.