Notch signaling plays a critical role in the maintenance of intestinal homeostasis. The aim of the present study was to investigate the role of Notch signaling in the apoptosis of intestinal epithelial cells after intestinal ischemia reperfusion (I/R) injury. Male C57BL/6 mice were subjected to sham operation or I/R injury. Intestinal tissue samples were collected at 12 h after reperfusion. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining showed that intestinal I/R injury induced significantly increased apoptosis of intestinal epithelial cells. Meanwhile, the mRNA expression of Jagged1, DLL1, Notch2, and Hes5, and protein expression of NICD2 and Hes5 were increased significantly after I/R injury in intestinal epithelial cells. In an in vitro IEC-6 culture model, flow cytometry analyses showed that inhibition of Notch signaling by γ-secretase inhibitor DAPT and the suppression of Hes5 expression using siRNA both significantly increased the apoptosis of IEC-6 cells under the condition of hypoxia/ reoxygenation (H/R). In conclusion, the Notch2/Hes5 signaling pathway was activated and involved in the regulation of intestinal epithelial cells apoptosis in intestinal I/R injury.