Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including in particular insulin and glucagon. However, its expression and biological role in the adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, both at the transcriptional and protein levels. More importantly, immunostaining analyses detected its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1-disruption. A similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human MEN1 glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the auto-regulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which are beyond the pancreatic endocrine cells.