Fundamentally understanding the suppressive mechanisms used by different subsets of tumor-infiltrating regulatory T (Treg) cells is critical for the development of effective strategies for antitumor immunotherapy. γδ Treg cells have recently been identified in human diseases including cancer. However, the suppressive mechanisms and functional regulations of this new subset of unconventional Treg cells are largely unknown. In the current studies, we explored the suppressive mechanism(s) used by breast tumor-derived γδ Treg cells on innate and adaptive immunity. We found that γδ Treg cells induced immunosenescence in the targeted naive and effector T cells, as well as dendritic cells (DCs). Furthermore, senescent T cells and DCs induced by γδ Treg cells had altered phenotypes and impaired functions and developed potent suppressive activities, further amplifying the immunosuppression mediated by γδ Treg cells. In addition, we demonstrated that manipulation of TLR8 signaling in γδ Treg cells can block γδ Treg-induced conversion of T cells and DCs into senescent cells in vitro and in vivo. Our studies identify the novel suppressive mechanism mediated by tumor-derived γδ Treg cells on innate and adaptive immunity, which should be critical for the development of strong and innovative approaches to reverse the tumor-suppressive microenvironment and improve effects of immunotherapy.