<p>Mesenteric ischemia frequently causes bowel necrosis even after recirculation, known as reperfusion injury, and no effective therapy has been validated for preserving the intestine. Adipose-derived mesenchymal stem cell-conditioned medium (MSC-CM), a tissue engineering and regenerative therapy, has been suggested as a feasible acute-phase treatment for organ damages. This study aimed to elucidate the therapeutic effects of MSC-CM on intestinal tissue injuries by ischemia and reperfusion. Mice were categorized into three groups that underwent either 60-min mesenteric artery occlusion (ischemia group), 60-min reperfusion following 60-min occlusion (reperfusion group), or ischemia–reperfusion with the same duration following intravenous administration of 200-µL MSC-CM by retroorbital injection (MSC-CM group). The distal ileum was harvested, and immunofluorescence staining with caspase-3 and LGR5, an intestinal stem cell-specific marker, was performed for evaluating the injury location and type of cells protected by MSC-CM. Moreover, LGR5, Notch1, Jagged 1, Hes1, DLL1, DLL3, and DLL4 mRNA expressions were measured using quantitative polymerase chain reaction. Ischemia extensively damaged the epithelial layer, and reperfusion-induced cellular apoptosis at the epithelial layer. MSC-CM administration was associated with preservation of cells at the crypt base, which were identified as intestinal stem cells using double-immunofluorescence staining. The MSC-CM group demonstrated significantly higher LGR5 expression than the reperfusion and ischemia groups. Similarly, the MSC-CM group exhibited higher Notch1 and Jag1 expressions, whereas the reperfusion group showed a higher Notch1 expression. In conclusion, MSC-CM use was associated with preservation of the intestinal stem cells at the crypt of villi and higher Notch1 and Jag1 expressions.</p>