Insulin deficiency drives the progression of both type 1 and type 2 diabetes. Pancreatic β-cell insulin expression and secretion are tightly regulated by nutrients and hormones; however, intracellular signaling proteins that mediate nutrient and hormonal regulation of insulin synthesis and secretion are not fully understood. SH2B1 is an SH2 domain-containing adaptor protein. It enhances the activation of the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription and the phosphatidylinositol 3-kinase pathways in response to a verity of hormones, growth factors, and cytokines. Here we identify SH2B1 as a new regulator of insulin expression. In rat INS-1 832/13 β-cells, SH2B1 knockdown decreased, whereas SH2B1 overexpression increased, both insulin expression and glucose-stimulated insulin secretion. SH2B1-deficent islets also had reduced insulin expression, insulin content, and glucose-stimulated insulin secretion. Heterozygous deletion of SH2B1 decreased pancreatic insulin content and plasma insulin levels in leptin-deficient ob/ob mice, thus exacerbating hyperglycemia and glucose intolerance. In addition, overexpression of JAK2 increased insulin promoter activity, and SH2B1 enhanced the ability of JAK2 to activate the insulin promoter. Overexpression of SH2B1 also increased the expression of Pdx1 and the recruitment of Pdx1 to the insulin promoter in INS-1 832/13 cells, whereas silencing of SH2B1 had the opposite effects. Consistently, Pdx1 expression was lower in SH2B1-deficient islets. These data suggest that the SH2B1 in β-cells promotes insulin synthesis and secretion at least in part by enhancing activation of JAK2 and/or Pdx1 pathways in response to hormonal and nutritional signals.