Pdx1, a β-cell-specific transcription factor, has been shown to play a crucial role in maintaining β-cell function through transactivation of β-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against β-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in β cells (βPdx1) and crossed these mice with Ins2 diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2 mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of βPdx1; Ins2 mice. The βPdx1; Ins2 mice exhibited significantly improved glucose tolerance, compared with control Ins2 littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that βPdx1; Ins2 mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of βPdx1; Ins2 mice. These findings suggest that the sustained expression of Pdx1 improves β-cell failure in Ins2 mice, at least partially through the preserving expression of β-cell-specific genes as well as improved localization of GLUT2.