Ionizing space radiation causes oxidative DNA damage and triggers oxidative stress responses, and compromised DNA repair mechanisms can lead to increased risk of carcinogenesis. Young adult mice with developed innate and adaptive immune systems that harbored either a conventional intestinal microbiota (CM) or an intestinal microbiota with a restricted microbial composition (RM) were irradiated with a total dose of 1 Gy delivered by high-energy protons (2.5 GeV/n, LET = 0.2–2 keV/μm) or silicon or iron ions (850 MeV/n, LET ≈ 50 keV/μm and 1 GeV/n, LET = 150 keV/μm, respectively). Six hours after whole-body irradiation, acute chromosomal DNA lesions were observed for RM mice but not CM mice. High-throughput rRNA gene sequencing of intestinal mucosal bacteria showed that Barnesiella intestinihominis and unclassified Bacterodiales were significantly more abundant in male RM mice than CM mice, and phylotype densities changed in irradiated mice. In addition, Helicobacter hepaticus and Bacteroides stercoris were higher in CM than RM mice. Elevated levels of persistently phosphorylated γ-H2AX were observed in RM mice exposed to high-energy protons compared to nonirradiated RM mice, and they also were associated with a decrease of the antioxidant glutathione in peripheral blood measured at four weeks after irradiation. After radiation exposure, CM mice showed lower levels of γ-H2AX phosphorylation than RM mice and an increase in specific RM-associated phylotypes, indicating a down-regulating force on DNA repair by differentially abundant phylotypes in RM versus a radiation-sensitive complex CM.