Radiation-induced brain injury is a common complication of brain irradiation that eventually leads to irreversible cognitive impairment. Evidence has shown that the gut microbiome may play an important role in radiation-induced cognitive function. However, the effects of gut microbiota on radiation-induced brain injury (RIBI) remain poorly understood. Here we studied the link between intestinal microbes and radiation-induced brain injury to further investigate the effects of intestinal bacteria on neuroinflammation and cognitive function. We first verified the differences in gut microbes between male and female mice and administered antibiotics to C57BL/6 male mice to deplete the gut flora and then expose mice to radiation. We found that depletion of intestinal flora after irradiation may act as a protective modulator against radiation-induced brain injury. Moreover, we found that pretreatment with depleted gut microbes in RIBI mice suppressed brain pro-inflammatory factor production, and high-throughput sequencing analysis of mouse feces at 1-month postirradiation revealed microbial differences. Interestingly, a proportion of Verrucomicrobia Akkermansia showed partial recovery. Additionally, short-chain fatty acid treatments increased neuroinflammation in the radiation-induced brain injury model. Although a further increase in cognitive function was not observed, brain injury was aggravated in whole-brain irradiated mice to some extent. The protective effects of depleted intestinal flora and the utilization of the brain-gut axis open new avenues for development of innovative therapeutic strategies for radiation-induced brain injury.