The intestinal crypt is a site of potential interactions between microbiota products, stem cells, and other cell types found in this niche, including Paneth cells, and thus offers a potential for commensal microbes to influence the host epithelium. However, the complexity of this microenvironment has been a challenge to deciphering the underlying mechanisms. We used in vitro cultured organoids of intestinal crypts from mice, reinforced with in vivo experiments, to examine the crypt-microbiota interface. We find that within the intestinal crypt, Lgr5+ stem cells constitutively express the cytosolic innate immune sensor Nod2 at levels much higher than in Paneth cells. Nod2 stimulation by its bona fide agonist, muramyl-dipeptide (MDP), a peptidoglycan motif common to all bacteria, triggers stem cell survival, which leads to a strong cytoprotection against oxidative stress-mediated cell death. Thus, gut epithelial restitution is Nod2 dependent and triggered by the presence of microbiota-derived molecules. Display omitted •The bacterial peptidoglycan motif MDP induces higher yield of intestinal organoids in vitro•Nod2 is expressed in Lgr5+ intestinal epithelial stem cells•Stem cell-expressed Nod2 is sufficient to provide cytoprotection•MDP protects stem cells from stress in vivo in a Nod2-dependent manner The intestinal crypt is the site of epithelial regeneration and of potential interactions between microbiota products and stem cells. Nigro et al. show that intestinal Lgr5+ stem cells express the cytosolic innate immune sensor Nod2, whose stimulation by the bacterial ligand muramyl-dipeptide affords potent cytoprotection to the stem cells.