Background <p>FOXO1 is known to act as a tumor suppressor gene in breast cancer, but its exact mechanism of action remains unclear.</p> Objective <p>This study aimed to clarify how FOXO1 suppresses breast cancer cell proliferation and induces apoptosis.</p> Methods <p>Breast cancer cell lines were generated with stable knockdown or overexpression of FOXO1. RT-qPCR and western blot assays were conducted to confirm transfection efficiency. CCK-8 and colony formation assays were used to assess cell proliferation, while flow cytometry measured apoptosis. The cells were subcutaneously injected into nude mice, and the volume and mass of the resulting tumors were evaluated. Immunohistochemistry was used to analyze Ki-67 expression in the tumors. A TUNEL assay examined apoptosis in the tumor cells. We performed bioinformatic analysis to identify FOXO1-targeted miRNAs and their downstream target mRNAs.</p> Results <p>Overexpression of FOXO1 inhibited breast cancer cell proliferation and promoted apoptosis. In contrast, knockdown of FOXO1 enhanced cell proliferation and reduced apoptosis. Among the downstream miRNAs we identified, miR-99a-5p was found to be downregulated in breast cancer tissue. FOXO1 binds to the miR-99a promoter, facilitating its transcription. Inhibition of miR-99a-5p partially reversed the effects of FOXO1 overexpression on cell proliferation and apoptosis. E2F7, a target mRNA of miR-99a-5p, showed a negative correlation with FOXO1 expression in breast cancer mRNAs we screened. Silencing E2F7 partially mitigated the inhibitory effects of miR-99a-5p on proliferation and apoptosis in FOXO1-overexpressing cells. E2F7 binds to the FOXO1 promoter, thus suppressing its transcription and reducing its expression.</p> Conclusion <p>FOXO1 suppresses breast cancer cell proliferation and promotes apoptosis by enhancing the transcription and expression of miR-99a-5p, while inhibiting its target gene E2F7. E2F7, in turn, represses the transcription of FOXO1, lowering its expression.</p>