The loss of pancreatic beta cell function leads to chronically high blood glucose levels, contributing to diabetes mellitus, one of the leading causes of morbidity and mortality worldwide. Understanding the molecular mechanisms that regulate beta cell function could pave the way for the development of more effective anti-diabetic treatments. In this study, we identify the evolutionarily conserved TSC22D1 protein as a novel regulator of beta cell function. TSC22D1 depletion in INS-1E cells enhances the expression of beta cell identity genes, including Ins1, Ins2, Pdx1, Slc2a2, and Nkx6.1 and promotes glucose-stimulated insulin secretion without altering intracellular insulin content. Mechanistically, TSC22D1 and FoxO1 interact and regulate each other reciprocally to control beta cell function. Our follow-up interactome analysis revealed other novel proteins that differentially bind to TSC22D1 depending on glucose availability. These include several RNA-binding proteins such as Hnrnpm and Ddx46, as well as distinct tubulin isoforms, chaperones, and Scgn, a well-known regulator of insulin secretion. To further investigate the impact of TSC22D1 deficiency in cellular response to glucose stimulation, we performed RNA sequencing (RNA-Seq) experiment. The RNA-Seq results strongly aligned with our mass spectrometry interactome data and revealed mRNA processing, ribonucleoprotein complex biogenesis, and Golgi vesicle transport within the top pathways that are enriched upon glucose stimulation in TSC22D1 deficient cells. Overall, our study confirms that beta cell function requires tight regulation of insulin synthesis and secretion at multiple levels, even by a single protein, which we identified as TSC22D1, further highlighting the potential of targeting TSC22D1 in the development of new therapeutic strategies for diabetes.