The large Maf family of basic leucine-zipper–containing transcription factors are known regulators of key developmental and functional processes in various cell types, including pancreatic islets. Here, we demonstrate that within the adult pancreas, MafB is only expressed in islet α-cells and contributes to cell type–specific expression of the glucagon gene through activation of a conserved control element found between nucleotides −77 to −51. MafB was also shown to be expressed in developing α- and β-cells as well as in proliferating hormone-negative cells during pancreatogenesis. In addition, MafB expression is maintained in the insulin⁺ and glucagon⁺ cells remaining in mice lacking either the Pax4 or Pax6 developmental regulators, implicating a potentially early role for MafB in gene regulation during islet cell development. These results indicate that MafB is not only important to islet α-cell function but may also be involved in regulating genes required in both endocrine α- and β-cell differentiation.