The intestinal hormone, glucagon-like peptide-2 (GLP-2), stimulates growth, survival, and function of the intestinal epithelium through increased crypt cell proliferation, and a long-acting analog has recently been approved to enhance intestinal capacity in patients with short bowel syndrome. The goal of the present study was to determine whether GLP-2-induced crypt cell proliferation requires a full complement of B-cell lymphoma Moloney murine leukemia virus insertion region-1 homolog (Bmi-1), using the Bmi-1eGFP/+ mouse model in comparison with age- and sex-matched Bmi-1+/+ littermates. Bmi-1 is a member of the polycomb-repressive complex family that promotes stem cell proliferation and self-renewal and is expressed by both stem cells and transit-amplifying (TA) cells in the crypt. The acute (6 h) and chronic (11 d) proliferative responses to long-acting human (Gly2)GLP-2 in the crypt TA zone, but not in the active or reserve stem cell zones, were both impaired by Bmi-1 haploinsufficiency. Similarly, GLP-2-induced crypt regeneration after 10-Gy irradiation was reduced in the Bmi-1eGFP/+ animals. Despite these findings, chronic GLP-2 treatment enhanced overall intestinal growth in the Bmi-1eGFP/+ mice, as demonstrated by increases in small intestinal weight per body weight and in the length of the crypt-villus axis, in association with decreased apoptosis and an adaptive increase in crypt epithelial cell migration rate. The results of these studies therefore demonstrate that a full complement of Bmi-1 is required for the intestinal proliferative effects of GLP-2 in both the physiological and pathological setting, and mediates, at least in part, the proliferation kinetics of cells in the TA zone.