BACKGROUND: Breast cancer is one of the leading causes of cancer mortality worldwide. 5-fluorouracil (5-FU) is one of the chemotherapy drugs to treat breast cancer, but it is associated with several side effects. Combination therapy is a way to increase the effectiveness of chemo drugs and decrease their usage dose. Quercetin (Quer) is one of the natural polyphenols with anti-cancer properties. This study investigated the apoptotic effect of 5-FU in combination with Quer compared with 5-FU alone on MCF-7 breast cancer cells. METHOD AND RESULTS: Different single and combined concentrations of 5-FU and Quer were applied to MCF 7 cells for 48 h. Cell viability, apoptosis, gene expression of Bax, Bcl2, and p53, caspase activity, and colony number were assessed using MTT assay, flow cytometry, quantitative real-time PCR, enzyme-linked immunosorbent (ELISA), and Colony formation assay, respectively. The combination of 5-FU and Quer compared to 5-FU alone improved apoptosis by increasing the gene expression of Bax and p53 and caspase-9 activity and decreasing the Bcl2 gene expression. Colony formation in MCF-7 cells significantly decreased in the combined state compared to 5-FU alone. CONCLUSION: Quer potentiates the sensitivity of breast cancer to 5-FU so that this combination may be proposed as a treatment for breast cancer. Therefore, this combination can be suggested for future in vivo studies.