The glucagon gene is expressed in α‐cells of the pancreas, L cells of the intestine and the hypothalamus. The determinants of the α‐cell‐specific expression of the glucagon gene are not fully characterized, although Arx, Pax6 and Foxa2 are critical for α‐cell differentiation and glucagon gene expression; in addition, the absence of the β‐cell‐specific transcription factors Pdx1, Pax4 and Nkx6.1 may allow for the glucagon gene to be expressed. Pax6, along with cMaf and MafB, binds to the DNA control element G₁ which confers α‐cell specificity to the promoter and to G₃ and potently activates glucagon gene transcription. In addition, to its direct role on the transcription of the glucagon gene, Pax6 controls several transcription factors involved in the activation of the glucagon gene such as cMaf, MafB and NeuroD1/Beta2 as well as different steps of glucagon biosynthesis and secretion. We conclude that Pax6 independently of Arx and Foxa2 is critical for α‐cell function by coordinating glucagon gene expression as well as glucagon biosynthesis and secretion.