Abstract: Objective: This study was to investigate the protective effect of Noni polysaccharides on ionizing radiation injury and its regulation of intestinal flora in mice. Methods: Fifty KM male mice were randomly divided into normal group, model group, noni polysaccharide low-dose group, noni polysaccharide medium-dose group and Noni polysaccharide high-dose group. The whole body of the mice was irradiated by the medical electron linear accelerator, thus establishing acute radiation-damaged mice model. Subsequently, survival quality of mice after irradiating was observed, and related biochemical indicators of serum and liver tissue of mice after irradiating for 72 h were detected. Furthermore, 16S rRNA sequencing of mouse feces was performed to analyze diversity of intestinal flora. Results: Noni polysaccharides could improve survival quality of radiation-damaged mice, and increase organ index, peripheral blood leukocyte (WBC) number, spleen nodule number, bone marrow DNA content. Moreover, noni polysaccharides could reduce the rate of bone marrow polychromatic erythrocyte micronucleus (MN-PCE), and increase the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum and liver, and reduce malondialdehyde (MDA) content. Compared with the MC group, the increase rate of peripheral white blood cell count and number of splenic noduleswas 46.85%, 38.23%, and the content of bone marrow DNA increased by 51.95%, the reduction rate of MN-PCE was 38.84% of Noni polysaccharide high-dose group. Meanwhile, the oxidative stress of mice after irradiating had improvement in different degree of Noni polysaccharide. Additionally, Noni polysaccharides could promote the growth of intestinal flora of radiation-damaged mice in a direction of beneficial to host health. Meanwhile, Noni polysaccharides could accelerate the growth of probiotics and inhibit the colonization of harmful bacteria, and recover the changes of intestinal flora caused by ionizing radiation. Conclusion: Noni polysaccharides exhibited obvious protective effect on ionizing radiation-damaged mice caused by X-ray, and showed certain restoring effect on intestinal flora disturbance caused by radiation.