Endogenous glucagon-like peptide-2 (GLP2) is a key mediator of refeeding-induced and resection-induced intestinal adaptive growth. This study investigated the potential role of GLP2 in mediating the mucosal responses to a chronic high-fat diet (HFD). In this view, the murine small intestine adaptive response to a HFD was analyzed and a possible involvement of endogenous GLP2 was verified using GLP2 (3–33) as GLP2 receptor (GLP2R) antagonist. In comparison with animals fed a standard diet, mice fed a HFD for 14 weeks exhibited an increase in crypt–villus mean height (duodenum, 27.5±3.0%; jejunum, 36.5±2.9%; P<0.01), in the cell number per villus (duodenum, 28.4±2.2%; jejunum, 32.0±2.9%; P<0.01), and in Ki67-positive cell number per crypt. No change in the percent of caspase-3-positive cell in the villus–crypt was observed. The chronic exposure to a HFD also caused a significant increase in GLP2 plasma levels and in GLP2R intestinal expression. Daily administration of GLP2 (3–33) (30–60 ng) for 4 weeks did not modify the crypt–villus height in control mice. In HFD-fed mice, chronic treatment with GLP2 (3–33) reduced the increase in crypt–villus height and in the cell number per villus through reduction of cell proliferation and increase in apoptosis. This study provides the first experimental evidence for a role of endogenous GLP2 in the intestinal adaptation to HFD in obese mice and for a dysregulation of the GLP2/GLP2R system after a prolonged HFD.