It is not clear whether different radiation methods have different effects on enamel. The purpose of this study was to compare the effects of single and fractionated radiation on enamel and caries susceptibility and to provide an experimental basis for further study of radiation‑related caries. Thirty-six caries-free human third molars were collected and randomly divided into three groups (n = 12). Group1(control group) was not exposed to radiation. Group 2 received single radiation with a cumulative dose of 70Gy. Group 3 underwent fractionated radiation, receiving 2Gy/day for 5days followed by a 2-day rest period, for a total of 7weeks with a cumulative dose of 70Gy. Changes in microhardness, roughness, surface morphology, bacterial adhesion and ability of acid resistance of each group were tested. Scanning electron microscope revealed that the enamel surface in both radiation groups exhibited unevenness and cracks. Compared with the control group, microhardness and acid resistance of enamel decreased, while roughness and bacterial adhesion increased in both the single radiation and fractionated radiation groups. Compared with the single radiation group, the enamel surface microhardness and acid resistance decreased in the fractionated radiation group, while roughness and bacterial adhesion increased. Both single radiation and fractionated radiation resulting in changes in the physical and biological properties of enamel, with these changes being more pronounced in the fractionated radiation group. Therefore, fractionated radiation is recommended as a more suitable method for constructing a radiation‑related caries model in vitro.