Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic β-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient β cells. To elucidate Foxo1's function in β cells, we produced a β-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a β-cell line from an insulinoma induced in this knockout mouse by the β-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced β-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to β-cell function. These cells should be useful for further studies on Foxo1's roles in β-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus.