MafB, a member of the large Maf transcription factor family, is essential for the embryonic and terminal differentiation of pancreatic α- and β-cells. However, the role of MafB in the control of adult islet-cell proliferation remains unknown. Considering its oncogenic potential in several other tissues, we investigated the possible alteration of its expression in adult mouse β-cells under different conditions of proliferation. We found that MafB, in general silenced in these cells, was reexpressed in ∼30% of adaptive β-cells both in gestational female mice and in mice fed with a high-fat diet. Importantly, reactivated MafB expression was also observed in the early β-cell lesions and insulinomas that developed in β-cell specific Men1 mutant mice, appearing in >80% of β-cells in hyperplasic or dysplastic islets from the mutant mice >4 months of age. Moreover, MafB expression could be induced by glucose stimulation in INS-1 rat insulinoma cells. The induction was further reinforced following Men1 knockdown by siRNA. Furthermore, MafB overexpression in cultured βTC3 cells enhanced cell foci formation both in culture medium and on soft agar, accompanied with the increased expression of Cyclin B1 and D2. Conversely, MafB downregulation by siRNA transfection reduced BrdU incorporation in INS-1E cells. Taken together, our data reveal that Men1 inactivation leads to MafB reexpression in mouse β-cells in vivo, and provides evidence that deregulated ectopic MafB expression may have a hitherto unknown role in adult β-cell proliferation and Men1-related tumorigenesis.