The Pax6 transcription factor is crucial for pancreatic α-cells. Indeed, Pax6-deficient mouse models are characterized by markedly altered α-cell differentiation. Our objective was to investigate the role of Pax6 in glucagon secretion process. We used a Pax6-deficient model in rat primary enriched-α cells with specific small interfering RNA leading to a 70% knockdown of Pax6 expression. We first showed that Pax6 knockdown decreases glucagon biosynthesis as well as glucagon release. Through physiological assays, we demonstrated that the decrease of Pax6 affects specifically acute glucagon secretion in primary α-cell in response to glucose, palmitate, and glucose-dependent insulinotropic peptide (GIP) but not the response to arginine and epinephrine. We identified in Pax6 knockdown model that genes involved in glucagon secretion such as the glucokinase (GCK), G protein-coupled receptor (GPR40), and GIP receptor (GIPR) as well as the corresponding proteins were significantly decreased whereas the insulin receptor (IR) Kir6.2/Sur1, and glucose transporter 1 genes were not affected. We demonstrated that Pax6 directly binds and activates specific elements on the promoter region of the GPR40, GCK, and GIPR genes. Finally, through site-directed mutagenesis experiments, we showed that disruption of Pax6 binding on the GCK, GPR40, and GIPR gene promoters led to specific decreases of their activities in the αTC1.9 glucagon-producing cell line. Hence our results indicate that Pax6 acts on the regulation of glucagon secretion at least through the transcriptional control of GCK, GPR40, and GIPR. We propose that Pax6 is not only critical for glucagon biosynthesis but also for glucagon secretion particularly in response to nutrients.