Purpose: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4,SMAD4) is involved in the adenoma–carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFβ pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced bySMAD4gene expression, as patients with decreasedSMAD4gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of theSMAD4andTGFB1genes. Patients and methods: The effect of 5-FU on the expression ofSMAD4andTGFB1in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. Results: Significant changes in the level ofSMAD4andTGFB1gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of theSMAD4gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of theSMAD4gene in CACO-2 cells. The level of expression of theTGFB1gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. Conclusion: The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affectSMAD4gene expression, which may increase the effectiveness of therapy.