Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcg^gfp/gfp mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcg^gfp/gfp mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcg^gfp/gfp mice were not significantly different from those in Gcg^+/+ and Gcg^gfp/+ mice, even after starvation for 16 h. Serum insulin levels in Gcg^gfp/gfp mice were lower than in Gcg^+/+ and Gcg^gfp/+ on ad libitum feeding, but no significant differences were observed on starvation. Islet α-cells and intestinal L-cells were readily visualized in Gcg^gfp/gfp and Gcg^gfp/+ mice under fluorescence. The Gcg^gfp/gfp postnatally developed hyperplasia of islet α-cells, whereas the population of intestinal L-cells was not increased. In the Gcg^gfp/gfp, expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of α-cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model.