Deficient insulin secretion and relative hyperproinsulinemia are characteristic of non–insulin-dependent diabetes. Culture of human islets at high glucose concentrations generates β-cell dysfunctions that mimic those observed in diabetic patients, with depleted islet insulin stores. The increased proinsulin ratio probably results from accelerated discharge of insulin granules, rather than diminished expression of convertase. Insulin mRNA levels are decreased, and the rate of transcription of the human insulin gene is suppressed. In human islets exposed to high glucose for 4 to 9 days, the DNA binding of PDX-1, the key transcriptional factor of the insulin gene, is reduced, as is its mRNA level. These effects are reversed by normalizing the culture glucose concentration. It is proposed that β-cell “glucotoxicity” results from the inability of proinsulin biosynthesis to keep pace with chronic insulin hypersecretion. At least in human β-cells, downregulation of PDX-1 expression by hyperglycemia is the main cause for depletion of the insulin stores.