Background: The small intestinal epithelium is highly sensitive to radiation and is a major site of injury during radiation therapy and environmental overexposure. Objective: To examine probiotic bacteria as potential radioprotective agents in the intestine. Methods: 8-week-old C57BL/6 wild-type or knockout mice were administered probiotic by gavage for 3 days before 12 Gy whole body radiation. The intestine was evaluated for cell-positional apoptosis (6 h) and crypt survival (84 h). Results: Gavage of 5×10 7 Lactobacillus rhamnosus GG (LGG) improved crypt survival about twofold (p<0.01); the effect was observed when administered before, but not after, radiation. Conditioned medium (CM) from LGG improved crypt survival (1.95-fold, p<0.01), and both LGG and LGG-CM reduced epithelial apoptosis particularly at the crypt base (33% to 18%, p<0.01). LGG was detected in the distal ileal contents after the gavage cycle, but did not lead to a detectable shift in bacterial family composition. The reduction in epithelial apoptosis and improved crypt survival offered by LGG was lost in MyD88 -/-, TLR-2 -/- and cyclo-oxygenase- 2 -/- (COX-2) mice but not TLR-4 -/- mice. LGG administration did not lead to increased jejunal COX-2 mRNA or prostaglandin E2 levels or a change in number of COX-2-expressing cells. However, a location shift was observed in constitutively COX-2-expressing cells of the lamina propria from the villi to a position near the crypt base (villi to crypt ratio 80:20 for control and 62:38 for LGG; p<0.001). Co-staining revealed these COX-2- expressing small intestinal lamina propria cells to be mesenchymal stem cells. Conclusions: LGG or its CM reduce radiation-induced epithelial injury and improve crypt survival. A TLR-2/ MyD88 signalling mechanism leading to repositioning of constitutive COX-2-expressing mesenchymal stem cells to the crypt base is invoked.