The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased beta and delta cells and absent alpha cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat alpha cells. To study the transcriptional network of Pax6 in adult and differentiated alpha cells, we generated Pax6-deficient primary rat alpha cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat alpha cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and alpha cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on alpha cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for alpha cell development. We conclude that Pax6 is critical for alpha cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and alpha cell differentiation.