Previous studies have demonstrated that (D‑Ala2, D‑Leu5)‑enkephalin (
DADLE) protects rats from hepatic
ischemia/reperfusion (I/R) injury. In the present study,
DADLE was also observed to alleviate IR‑induced intestinal epithelial cell injury in rats by inhibiting mitogen‑activated
protein kinase kinase 7 (MKK7)‑c‑Jun N‑terminal
kinase (JNK) pathway signaling. To investigate the protective effect of
DADLE on
hypoxia/reoxygenation injury in rat intestinal epithelial cells, rat intestinal epithelial cells were treated with different concentrations of
DADLE, following which the cell survival rate was determined using a tetrazolium (MTT) colorimetric assay, and apoptosis was determined using flow cytometry. To confirm whether the protective effect of
DADLE was due to its effect on MKK7‑JNK signaling, the phosphorylation levels of MKK7 and JNK were analyzed using western blot analysis following treatment with different concentrations of
DADLE. The results demonstrated that, following treatment with
DADLE, the survival rate of the rat intestinal cells subjected to I/R‑induced injury increased significantly and the apoptotic rate decreased in a concentration‑dependent manner. In addition, the levels of phosphorylated MKK7 and JNK decreased in a concentration‑dependent manner following treatment with
DADLE. Silencing the gene expression of MKK7 using
small interfering RNA prior to
DADLE treatment resulted in a reduction in the protective effects of
DADLE on the rat intestinal epithelial cells subjected to I/R injury. Collectively, the results of the present study demonstrated that the protective effects of
DADLE in I/R injury in rat intestinal cells occurred through inhibition of the MKK7‑JNK pathway.