Intestinal injury is one of the major side effects that are induced by medical radiation exposure, and has limited effective therapies. In this study, we investigated the beneficial effects of sanguinarine (SAN) on intestinal injury induced by ionizing radiation (IR) both in vitro and in vivo. Mice were exposed to whole abdominal irradiation (WAI) to mimic clinical scenarios. SAN was injected intraperitoneally to mitigate IR-induced injury. Histological examination was performed to assess the tissue injuries of the spleen and small intestine. A small intestinal epithelial cell line-6 (IEC-6) was analyzed for its viability and apoptosis in vitro under different treatments. Inflammation-related pathways and serum inflammatory cytokines were detected via Western blot analysis and ELISA, respectively. High-throughput sequencing was used to characterize the gut microbiota profile. High-performance liquid chromatography was performed to assess short-chain fatty acid contents in the colon. In vitro, SAN pretreatment protected cell viability and reduced apoptosis in IEC-6 cells. In vivo, SAN pretreatment protected immune organs, alleviated intestinal injury, and promoted intestinal recovery. SAN also reduced the levels of inflammatory cytokines, suppressed high mobility group box 1 (HMGB1)/ Toll-like receptor 4 (TLR4) pathway activation, and modulated gut microbiota composition. Our findings demonstrate that the beneficial properties of SAN alleviated intestinal radiation injury. Thus, SAN represents a therapeutic option for protecting against IR-induced intestinal injury in preclinical settings. Display omitted •SAN protected radiation-induced intestinal injury by downregulation of HMGB1/TLR4 pathway and reduction of proinflammatory cytokines.•SAN regulated composition and function of gut microbiota in irradiated mice.•SAN is a promising therapeutic option for treatment of adverse side effects of radiation exposure.