PURPOSE: Fanconi anemia complementation group F (FANCF) is a key factor to maintaining the function of Fanconi anaemia/BRCA (FA/BRCA) pathway, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In the present study, we evaluated the chemosensitization effect of FANCF in breast cancer cells. METHODS: We performed specific knockdown of the endogenous FANCF in breast cancer cells by transfecting the cells with an FANCF short hairpin RNA (shRNA) vector. Cell viability was measured with a Cell Counting Kit-8, and DNA damage was assessed with the alkaline comet assay. The apoptosis, cell cycle, and drug accumulation were measured by flow cytometric analysis. Protein expression levels were determined by Western blot analysis, using specific antibodies. RESULTS: The analyses of two breast cancer cell lines (MCF-7 and MDA-MB-435S) demonstrated that the FANCF shRNA could effectively block the FA/BRCA pathway through the inhibition of Fanconi anemia complementation group D2 ubiquitination. Moreover, FANCF silencing potentiated the sensitivity of cells to mitomycin C (MMC), where combined FANCF shRNA/MMC treatment inhibited cell proliferation, induced S-phase arrest, apoptosis, and DNA fragmentation, and reduced the mitochondrial membrane potential, compared with MMC treatment alone. CONCLUSION: Taken together, this study demonstrates that the inhibition of FANCF by its shRNA leads to a synergistic enhancement of MMC cytotoxicity in breast cancer cells. These results suggest that the inhibition of the FA/BRCA pathway is a useful adjunct to cytotoxic chemotherapy for the treatment of breast cancer.