Altered beta cell mass and/or dysfunction are central pathophysiological features in type 2 diabetes (T2D). Recent genetic findings have highlighted the transcription factor GLIS3 as a susceptibility gene for T2D, neonatal diabetes and congenital hyperthyroidism. GLIS3 maps to a locus associated with diabetes phenotypes in a genetic cross derived from the Goto-Kakizaki (GK) rat model of spontaneous T2D and Brown Norway (BN) controls. We identified DNA variants in Glis3 in the GK, including two non synonymous variants leading to potentially functionally important amino acid substitutions. We developed a congenic strain (CS1.a) which carries a short (1.7MB) GK genomic region containing Glis3 and surrounding genes (including Slc1a1), isolated on a BN resistant background, to test the relevance of these variants to diabetes, and carried out functional studies to identify GLIS3 targets. We determined the genomic sequence across the entire GK haplotype in the CS1.a congenics. Glucose tolerance and insulin secretion tests were performed in CS1.a and BN rats. For transfection experiments, the promoter region of Slc1a1 was cloned into pGL3 basic vector upstream of the luciferase reporter gene, and co-transfected into INS-1 cells, together with the expression vector pcDNA3 containing the full cDNA for Glis3. A GLIS3 antibody (Abcam) was used for chromatin immunoprecipitation. Rats of the CS1-a strain exhibited hyperglycaemia and increased glucose-induced insulin secretion in vivo and in vitro. In INS1 cells, glucose induced insulin secretion was significantly altered when cells were transfected with a plasmid containing the GK allele of GLIS3. Sequence analysis of the GK congenic region identified polymorphisms in the promoter region of Slc1a1, close to a common binding motif for GLIS3. Luciferase assay of the cloned Slc1a1 promoter region showed that the GK variants are functional and are associated with decreased promoter activity. Results from chromatin immunoprecipitation experiments in INS1 cells showed an enrichment of SLC1A1 when GLIS3 was immunoprecipitated. Cotransfection experiments in INS1 cells demonstrated the interaction of GLIS3 with the promoter of Slc1a1, which could be further confirmed by ChIP-seq experiments. We showed the impact of GK polymorphisms in GLIS3 on insulin secretion, which may at least partly account for the diabetes locus identified in the GKxBN cross. Our results demonstrate the functional impact of GK polymorphisms identified in Slc1a1 promoter and suggest that the gene encoding the glutamate transporter Slc1a1 is a direct target of GLIS3. These results provide advances in the characterization of the role of GLIS3 on insulin secretion and the molecular mechanisms involved.