Thymidine phosphorylase (dThdPase) is an enzyme that converts 5′‐deoxy‐5‐fluorouridine (5′DFUR) to the toxic substance 5‐fluorouracil (5‐FU); it is also known to be a platelet‐derived endothelial cell growth factor. In order to investigate the feasibility of suicide gene therapy against colorectal cancer by means of the combination of 5′DFUR and the converting enzyme dThdPase, we transfected the dThdPase gene into the human colon cancer cell line SW480 and analyzed the growth pattern as well as the sensitivity to 5‐FU or 5′DFUR of the dThdPase‐transfected cells. The 50% inhibition (IC50) values of 5‐FU against the SW480 parental cells, control vector‐transfected cells SW480/V1, and dThdPase‐transfected cells SW480/dThdPase were approximately 4.9, 6.3, and 2.9 μM, respectively. The IC50 of SW480/dThdPase was lower than that of SW480 or SW480/V1, although the differences were not statistically significant. The IC50 values of 5′DFUR for SW480, SW480/V1, and SW480/dThdPase were approximately 300, 330, and 3.2 αM, respectively. The sensitivity to 5′DFUR of SW480/dThdPase was increased by about 100‐fold compared with that of SW480 or SW480/V1. With only 10% transfection efficacy, a high enough sensitivity to 5′DFUR was obtained to suppress the cell growth, indicating that a strong bystander effect was induced by this system. The in vivo growth of the s.c. transplanted SW480/dThdPase tumor in nude mice was significantly suppressed by i.p. injection of 5′DFUR compared with that in control mice that received phosphate‐buffered saline (PBS) treatment. These results suggest that gene therapy using the combination of 5′DFUR and the dThdPase gene may be a useful approach for treatment of colon cancer.