Background and Purpose The colonic epithelium is highly sensitive to ionizing radiation, leading to impaired barrier function. Lysophosphatidic acid (LPA) is protective against radiation-induced intestinal mucosal injury and genotoxic stress. We evaluated the effect of LPA and its analog, Radioprotein-1 in radiation-induced colonic epithelial barrier dysfunction. Experimental approach Caco-2 and m-ICC12 cell monolayers were exposed up to g-radiation, and the barrier function was evaluated by measuring and unidirectional flux of FITC-inulin. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was analyzed by evaluating mucosal permeability to inulin and measuring plasma lipopolysaccharide (LPS) levels. Tight junction and adherens junction integrity was examined by confocal microscopy. Oxidative stress was assessed by measuring protein thiol oxidation and antioxidant mRNA. Key Results In Caco-2 and m-ICC12 cell monolayers, LPA attenuates radiation-induced redistribution of tight junction proteins from the junctions, which was blocked by Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupt colonic epithelial tight junction and adherens junction, increases mucosal inulin permeability and elevates plasma LPS. RP1 administered 30 min pre-irradiation or 24 hours post-irradiation alleviates TBI and PBI-BM5-induced tight junction disruption, barrier dysfunction, and endotoxemia. The RP1 effects on radiation-induced colonic injury was associated with protein thiol oxidation, suppression of antioxidant gene expression, cofilin activation and remodeling of actin cytoskeleton. Conclusion and Implications These data demonstrate that LPA2 receptor agonists prevent and mitigate g-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia, indicating their potential therapeutic benefit in the treatment of the gastrointestinal acute radiation syndrome.