The MafA transcription factor is both critical to islet β-cell function and has a unique pancreatic cell-type-specific expression pattern. To localize the potential transcriptional regulatory region (s) involved in directing expression to the β cell, areas of identity within the 5′ flanking region of the mouse, human, and rat mafA genes were found between nucleotides− 9389 and− 9194,− 8426 and− 8293,− 8118 and− 7750,− 6622 and− 6441,− 6217 and− 6031, and− 250 and+ 56 relative to the transcription start site. The identity between species was greater than 75%, with the highest found between bp− 8118 and− 7750 (∼ 94%, termed region 3). Region 3 was the only upstream mammalian conserved region found in chicken mafA (88% identity). In addition, region 3 uniquely displayed β-cell-specific activity in cell-line-based reporter assays. Important regulators of β-cell formation and function, PDX-1, FoxA2, and Nkx2. 2, were shown to specifically bind to region 3 in vivo using the chromatin immunoprecipitation assay. Mutational and functional analyses demonstrated that FoxA2 (bp− 7943 to− 7910), Nkx2. 2 (bp− 7771 to− 7746), and PDX-1 (bp− 8087 to− 8063) mediated region 3 activation. Consistent with a role in transcription, small interfering RNA-mediated knockdown of PDX-1 led to decreased mafA mRNA production in INS-1-derived β-cell lines (832/13 and 832/3), while MafA expression was undetected in the pancreatic epithelium of Nkx2. 2 null animals. These results suggest that β-cell-type-specific mafA transcription is principally controlled by region 3-acting transcription factors that are essential in the formation of functional β cells.