The increasing use of radiation presents a risk of radiation exposure, making the development of radioprotectors necessary. In the previous study, it is investigated that Deinococcus radiodurans (R1‐EVs) exert the antioxidative properties. However, the radioprotective activity of R1‐EVs remains unclear. In the present study, the protective effects of R1‐EVs against total body irradiation (TBI)‐induced acute radiation syndrome (ARS) are investigated. To assess R1‐EVs' radioprotective efficacy, ARS is induced in mice with 8 Gy of TBI, and protection against hematopoietic (H)‐ and gastrointestinal (GI)‐ARS is evaluated. The survival rate of irradiated mice group decreases substantially after irradiation. In contrast, pretreatment with R1‐EVs increases the survival rates of the mice. The administration of R1‐EVs provides effective protection against radiation‐induced death of bone marrow cells and splenocytes by scavenging reactive oxygen species (ROS). Additionally, R1‐EVs protect both intestinal stem and epithelial cells from radiation‐induced apoptosis. R1‐EVs stimulate the production of short‐chain fatty acids in the gastrointestinal tract, suppress proinflammatory cytokines, and increase regulatory T cells in pretreated mice versus the irradiation‐only group. Proteomic analysis shows that the R1‐EV proteome is significantly enriched with proteins involved in oxidative stress response. These findings highlight R1‐EVs as potent radioprotectors with applications against radiation damage and ROS‐mediated diseases.