There are increasing reports showing the clinical significance of the p53 polymorphism status in terms of the response to chemotherapy. We investigated whether p53 polymorphism and mutation were associated with in vitro sensitivity to 5-fluorouracil (5-FU) in patients with colorectal cancer. Chemosensitivity to 5-FU was evaluated by the collagen gel droplet embedded culture drug sensitivity test. 5-FU sensitivity of tumor cells without inactive p53 mutation in the arginine/arginine (Arg/Arg) variant was significantly higher than that of tumor cells with or without inactive p53 mutation in other variants (p = 0.022), whereas the 5-FU sensitivity of tumor cells with inactive p53 mutation in the Arg/Arg variant was significantly lower than that of tumor cells with or without inactive p53 mutation in other variants (p = 0.002). In the Arg/Arg variant, apoptotic cells induced by 5-FU treatment in patients without inactive p53 mutation were more markedly increased than those in patients with inactive p53 mutation (p = 0.037). Bax and Bcl-2 protein expressions in tumor tissue treated with 5-FU were associated with both 5-FU sensitivity and the apoptotic cell count. Our data show that the Arg/Arg genotype without inactive p53 mutation could be predictive of a more favorable response and the Arg/Arg genotype with inactive p53 mutation a less favorable response to chemotherapy using 5-FU in CRC. The combination of the p53 codon 72 polymorphism and p53 mutation status is a potential predictive marker of sensitivity to 5-FU in CRC.