OBJECTIVE This study intended to observe the effects of methoxyamine (Mx) on cytotoxic effects and DNA damage caused by 5-Fluorouracil (5-FU) in combination with gamma radiation in a human colon cancer cell line, HT29. METHODS In this experimental study, HT29 cells were cultured as a monolayer and treated with different concentrations of 5-FU along with 1 mM Mx for 24 hours. Next, the cells were irradiated with 2 Gy gamma radiation. After the treatments, we assessed for DNA damage, cytotoxicity, and viability by alkaline comet, clonogenic survival, and trypan blue dye exclusion assays. RESULTS Cytotoxicity and DNA damage increased with increasing 5-FU concentration. The 1 mM Mx concentration had no significant effect on cytotoxicity and DNA damage from 5-FU; however, it increased the cytotoxic and genotoxic effects of different concentrations of 5-FU when used in combination with 2 Gy gamma radiation. CONCLUSIONS Mx combined with 5-FU enhanced the radiosensitivity of colon cancer cells.