Over-nutrition and a sedentary lifestyle are associated with increased intestinal permeability. This condition promotes obesity and associated metabolic disorders. Sestrin2 (SESN2) is a stress-inducible protein thought to promote the survival and recovery of epithelial cells and act as a positive regulator in exercise-induced improvements of glycolipid metabolism. Here we aimed to test the hypothesis that chronic exercise can protect intestinal barrier function against high-fat diet induced permeabilization through SESN2. WT and SESN2−/− mice were randomly assigned to five groups, fed with either normal chow or high fat diet (HFD), and provided with or without exercise training for 15-week. Metabolic parameters, fecal microbiota composition, and intestinal barrier integrity were assessed. The role of the gut microbiota was investigated by administering a mixture of broad-spectrum antibiotics (ABX). Fifteen-week HFD feeding induced dysmetabolism, dysbiosis and gut barrier dysfunctions in the WT mice. These effects were exaggerated in SESN2−/− mice. Chronic aerobic exercise significantly reversed HFD-induced pathologic changes, while SESN2 ablation weakened the protective effects of exercise. ABX did not abolish the differences in gut barrier function between WT and SESN2−/− mice. We speculated that SESN2 may protect intestinal integrity partly independent of gut microbiome. Combining ex vivo and in vivo approaches, we demonstrated that SESN2/pAMPK-Thr172/HIF-1α pathway may play an important role in exercise- improved intestinal permeability. Taken together, our study demonstrated that HFD and SESN2-KO have synergistic effects on intestinal homeostasis. SESN2 is crucial in exercise-improved intestinal permeability.