Glucagon-like peptide 1 (GLP-1) can increase pancreatic β-cells, and α-cells could be a source for new β-cell generation. We investigated whether GLP-1 increases β-cells through α-cell transdifferentiation. New β-cells originating from non-β-cells were significantly increased in recombinant adenovirus expressing GLP-1 (rAd-GLP-1)-treated RIP-CreER;R26-YFP mice. Proliferating α-cells were increased in islets of rAd-GLP-1-treated mice and αTC1 clone 9 (αTC1-9) cells treated with exendin-4, a GLP-1 receptor agonist. Insulinglucagon cells were significantly increased by rAd-GLP-1 or exendin-4 treatment in vivo and in vitro. Lineage tracing to label the glucagon-producing α-cells showed a higher proportion of regenerated β-cells from α-cells in rAd-GLP-1-treated Glucagon-rtTA;Tet--Cre;R26-YFP mice than rAd producing β-galactosidase-treated mice. In addition, exendin-4 increased the expression and secretion of fibroblast growth factor 21 (FGF21) in αTC1-9 cells and β-cell-ablated islets. FGF21 treatment of β-cell-ablated islets increased the expression of pancreatic and duodenal homeobox-1 and neurogenin-3 and significantly increased insulinglucagon cells. Generation of insulinglucagon cells by exendin-4 was significantly reduced in islets transfected with FGF21 small interfering RNA or islets of FGF21 knockout mice. Generation of insulin cells by rAd-GLP-1 treatment was significantly reduced in FGF21 knockout mice compared with wild-type mice. We suggest that GLP-1 has an important role in α-cell transdifferentiation to generate new β-cells, which might be mediated, in part, by FGF21 induction.