One of the most common malignancies in women is breast cancer. β-escin has pharmacological anticancer effects. 5-fluorouracil (5-FU) has antimetabolite and antiproliferative properties. The purpose of this study was to investigate the combined effects of 5-FU and β-escin on apoptosis, colony formation, Bcl-2 signaling protein, and p53 gene expression in MCF7 breast cancer cell line. The cytotoxic effects, the number of colonies, apoptosis, p53 gene expression, and Bcl-2 signaling protein of the combined 5-FU and β-escin on MCF7 cells were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic assay, flow cytometry, real-time quantitative polymerase chain reaction, and western blotting methods, respectively. Half-maximal inhibitory concentration values of β-escin and 5-FU were 80 μg/ml and 2 μM, respectively. The combination of 5-FU and β-escin on MCF7 cell viability showed a combination index equal to 0.5. The expression of p53 and apoptosis increased in the combination of 5-FU and β-escin on MCF7 cells compared to that of control group (P < 0.05). In addition, the number of colonies and Bcl-2 signaling protein in combination of 5-FU and β-escin decreased with respect to untreated control cells or single treatment of 5-FU and β-escin. The combination of 5-FU and β-escin not only has synergistic effects by increasing cell apoptosis and p53 gene expression but also decreases Bcl-2 signaling protein in MCF7 cell lines.