FOXP3 (forkhead box P3: also known as IPEX, XPID) is not only a hallmark of immunosuppressive regulatory T cells (Tregs), but also an X-linked breast cancer suppressor gene expressed in tumor cells. A two-stage investigation was conducted in individuals from northern, southern and eastern China. Individuals carrying a FOXP3 rs2294021CT genotype showed about 1.5-fold increased risk of breast cancer compared with TT carriers. In a related biochemical assay, the rs2294021C allele was found to significantly enhance transcription activity, leading to higher mRNA levels of FOXP3 compared with T allele. Moreover, the number of Tregs and its corresponding interleukin-10 (IL-10) secretion were elevated whereas the proliferation of antitumor T cells was decreased in the C-allele carriers. The breast cancer oncogenes Her-2/ErbB2 and Skp2 were also found to be significantly inhibited in C-allele carriers. Moreover, skewed X-chromosome inactivation (SXCI) analysis showed that rs2294021CT carriers with SXCI showed higher risk than the homozygous carriers and CT carriers without SXCI, suggesting a possible interaction between the rs2294021CT genotype and SXCI. Taken together, these findings indicate that the rs2294021CT genotype may increase an individual's susceptibility to breast cancer by breaking the balance between Treg-mediated immune tolerance and FOXP3-controlled tumor-suppressive effect.