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Some flavonoids have been shown to exhibitgood antioxidant activity and protect mice from damageinduced by radiation. Amentoflavone (AMF), a biflavonoidderived from the traditional herb-Selaginella tamariscina,has been reported to have antioxidant properties. The protectiveeffects and mechanism of action of AMF against radiationinjury remain unknown. In this study, male C57BL/6mice were subjected to total-body 60Co γ-irradiation at 7.5or 3.0 Gy. The survival rate and mean survival time wereevaluated to determine the radioprotective effect of AMF. Number of peripheral blood cells, frequency of colony formingunit-granulocytes, monocytes and micronuclei weremeasured to assess the protective effects of AMF on thehematopoietic system. Levels of superoxide dismutase andglutathione, and pathological changes in the bone marrowwere determined. Additionally, next-generation sequencingtechnology was used to explore potential targets of AMF. Weobserved that AMF markedly extends average survival time,reduces injury to the hematopoietic system and promotes itsrecovery. Furthermore, treatment with AMF significantlyattenuated radiation-induced oxidative stress. In addition,AMF had a significant effect on gene tumor necrosis factor alpha-induced protein 2. Together, the results of this studysuggest that AMF is a potential protective agent againstradiation injury.