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Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules. To assess crypt survival, a clonogenic microcolony assay was performed with 15 Gy of X-ray irradiation. The fractional crypt survival rate (46.0 &#177; 15.5% vs. 24.7 &#177; 9.2%, <i>p</i> &lt; 0.01) and fractional EdU-positive crypt survival rate (29.8 &#177; 14.5% vs. 9.79 &#177; 4.37%, <i>p</i> = 0.015) were significantly decreased in the NOD2^{&#8722;/&#8722;} mice compared with the wild-type (WT) mice at 3.5 days after irradiation. To evaluate intestinal epithelial regeneration capability, organoid reconstitution assays were performed. Small bowel crypts of the WT and NOD2^{&#8722;/&#8722;} mice were isolated and seeded into Matrigel for 3D culture. In the organoid reconstitution assays, the number of organoids formed did not differ between the NOD2^{&#8722;/&#8722;} and WT mice. Organoid formation ability was also assessed after exposure to 5 Gy irradiation. Organoid formation ability was significantly decreased in the NOD2^{&#8722;/&#8722;} mice compared with the WT ones after exposure to 5 Gy irradiation (33.2 &#177; 5.9 vs. 19.7 &#177; 8.8/well, <i>p</i> &lt; 0.01). NOD2 supports crypt survival after potentially lethal irradiation damage and is associated with intestinal epithelial regeneration.