Therefore, the aim of the present study was to evaluate the potential of 5-FU in treating late-stage disease by determining the effect of 5-FU on the viability of advanced colorectal cancer cell lines at different concentrations and whether 5-FU may influence SMAD4 gene expression, which is a prognostic factor in patients with colorectal cancer, and it also plays an important role in the response to 5-FU treatment. Here, SMAD4 is linked to colorectal cancer.