Abstract Understanding the role of different subtypes of tumor-infiltrating lymphocytes (TILs) in the tumor immunosuppressive microenvironment is essential to cancer treatment and elimination. We recently discovered that enriched gammadelta1 T cell populations in breast cancer TILs can suppress naïve and effector T cell responses and block the maturation and activities of dendritic cells. However, the mechanism(s) governing the gammadelta regulatory T (Treg) cell increase in breast cancer patients remains elusive. In this study, we showed that IP-10 secreted by breast cancer cells significantly attracted the migration of gammadelta Treg cells. Using the loss-of-function assay with neutralizing antibodies against the chemokines secreted by breast cancer cells, we demonstrated that IP-10 was the only functional chemokine that attracts the migration of gammadelta Treg cells by breast cancer cells. We further demonstrated that human breast tumor cells also can attract gammadelta Treg cells in vivo in NOD-SCID mice using live cell imaging systems. In addition, neutralization of IP-10 and CXCR3 significantly inhibited the migration and trafficking of gammadelta Treg cells to the breast tumor sites in vivo. These studies identify the mechanism responsible for the accumulation of gammadelta Treg cells in breast cancer patients and provide novel immunologic approaches capable of preventing the trafficking and generation of gammadelta Treg cells in breast tumor suppressive microenvironments.