Six million cancer patients worldwide receive radiotherapy, either alone or in combination with other cancer treatments annually. The purpose of this study was to investigate, at the structural and molecular levels, the protective effect of N-acetylcysteine (NAC), a powerful antioxidant, against radiotherapy-induced damage on the intestinal system. Three study groups were constituted: control (group 1: no radiotherapy), radiotherapy (RT; group 2), and RT + NAC (group 3). The NAC group received 300 mg/kg NAC orally for five days before irradiation. At the end of the 5th day, 6 Gy was irradiated once, and 300 mg/kg NAC was administered orally for two days following irradiation. NAC drug administration was maintained for a total of seven days. At the end of the study, the rats were euthanized by the administration of anesthetic agents and sacrificed. The malondialdehyde (MDA) levels and caspase-3 expression increased and glutathione (GSH) levels decreased in RT rats compared to the control group (p < 0.05). NAC reduced MDA levels, and caspase-3 expression, and increased GSH levels in the small intestine caused by RT (p < 0.05). Therefore, NAC may be useful in the prevention of gastrointestinal syndrome in patients undergoing radiotherapy in the treatment of malignancy. Impact statement Some six million cancer patients currently receive radiotherapy. Radiotherapy eliminates cancer cells by accelerating their death. However, radiotherapy is not selective, and it therefore harms healthy tissues around cancerous tissue. The latest studies have shown that the irradiation of biological materials causes a rapid increase in reactive oxygen species (ROS) in the tissue as a result of exposure of the target molecule to direct and indirect ionization. N-acetylcysteine (NAC) is an antioxidant that permits the elimination of free oxygen radicals and that contributes to glutathione synthesis. Our study, therefore, examined the effects of radiation resulting from radiotherapy on the small intestine at the molecular level, and prospectively considered the potential protective characteristics of NAC against gastrointestinal syndrome resulting from radiotherapy.