CCR6(+) subset of CD4(+) regulatory T cells, a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6(+) Tregs are present in tumor environment and their relation to the prognosis of tumor remain to be elucidated. In this study, we found that CCR6(+) CD4(+) CD25(high) Tregs, expressing high levels of CD45RO, are dominantly enriched in tumor mass from patients with breast cancer. Furthermore, the frequency of CCR6(+) Tregs, but not CCR6(-) Tregs in tumor infiltrating lymphocytes (TILs), significantly increased in patients during tumor progression, which reversely correlated with decreased frequency of the IFN-gamma(+)CD8(+)T cells in TILs. Most importantly, the frequency of CCR6(+) Tregs, but not CCR6(-) Tregs, reversely correlated to the survival of patients with breast cancer. This study suggested that a new subset of tumor-resident Tregs, CCR6(+) Tregs, may be dominantly responsible for the immunosuppression in tumor immunity and a potential predictor of the poor prognosis of breast cancer.