Our study demonstrates that 5-Fluorouracil induces a robust senescence program in both normal and P53-competent colorectal cancer cells, characterized by classical markers such as increased SA-β-galactosidase activity, mitochondrial depolarization, oxidative stress, AIF upregulation, and upregulation of P16, P21, and P53. This evidence concerns the gene TP53 and colorectal cancer.