The Pax6 transcription factor is crucial for endocrine cell differentiation and function. Indeed, mutations of Pax6 are associated with a diabetic phenotype and a drastic decrease of insulin-positive cell number. Our aim was to better define the β-cell Pax6 transcriptional network and thus provide further information concerning the role of Pax6 in β-cell function. We developed a Pax6-deficient model in rat primary β-cells with specific small interfering RNA leading to a 75% knockdown of Pax6 expression. Through candidate gene approach, we confirmed that Pax6 controls the mRNA levels of the insulin 1 and 2, Pdx1, MafA, GLUT2, and PC1/3 genes in β-cells. Importantly, we identified new Pax6 target genes coding for GK, Nkx6.1, cMaf, PC2, GLP-1R and GIPR which are all involved in β-cell function. Furthermore, we demonstrated that Pax6 directly binds and activates specific elements on the promoter region of these genes. We also demonstrated that Pax6 knockdown led to decreases in insulin cell content, in insulin processing, and a specific defect of glucose-induced insulin secretion as well as a significant reduction of GLP-1 action in primary β-cells. Our results strongly suggest that Pax6 is crucial for β-cells through transcriptional control of key genes coding for proteins that are involved in insulin biosynthesis and secretion as well as glucose and incretin actions on β-cells. We provide further evidence that Pax6 represents a key element of mature β-cell function.