Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of the steroid hormones estrogen and progesterone. Forkhead box A1 (FOXA1) is a member of the forkhead box transcription factor family and functions as a pioneer factor of the estrogen receptor (ER) in breast cancer. In the present study, we demonstrate that FOXA1 mRNA was upregulated by estrogen and that estrogen receptor-α (ERα) recruitment to ER-binding sites in the vicinity of the FOXA1 gene was increased by estrogen in ERα-positive MCF-7 breast cancer cells. The estrogen-induced FOXA1 upregulation was repressed by 4-hydroxytamoxifen treatment. We also demonstrated that the proliferation and the migration of MCF-7 cells were decreased by FOXA1-specific small interfering RNA (siRNA; siFOXA1). Furthermore, siFOXA1 decreased the estrogen response element-driven transcription and the estrogen-dependent upregulation of ERα target genes in MCF-7 cells. Next, the immunohistochemical analyses of FOXA1 were performed using two groups of breast cancer specimens. The nuclear immunoreactivity of FOXA1 was detected in 80 (74%) of 108 human invasive breast cancers and was negatively correlated with tumor grade and positively correlated with hormone receptor status, including ERα and progesterone receptor, pathological tumor size, and immunoreactivity of FOXP1, another FOX family transcription factor. FOXA1 immunoreactivity was significantly elevated in the relapse-free breast cancer patients treated with tamoxifen. Notably, the double-positive immunoreactivities of FOXA1 and FOXP1 were significantly associated with a favorable prognosis for the relapse-free and overall survival of patients with tamoxifen-treated breast cancer, with lower P values compared with FOXA1 or FOXP1 immunoreactivity alone. These results suggest that FOXA1 plays an important role in the proliferation and migration of breast cancer cells by modulating estrogen signaling and that the double-positive immunoreactivities of FOXA1 and FOXP1 are associated with a favorable prognosis of tamoxifen-treated breast cancer.