Failure of pancreatic β-cells contributes to the development of type 2 diabetes. Besides evidence of reduced glucose-stimulated insulin secretion and β-cell mass, little information is available about the molecular deficits of human diabetic islets. Islets were isolated from macroscopically normal pancreatic tissue from 8 patients with type 2 diabetes and 17 matched non-diabetic patients who underwent pancreatic surgery. Insulin content and insulin secretion were measured before and after islet stimulation with 25 mM glucose for 2 hours. In parallel, we also investigated the subcellular localization of polypyrimidine tract-binding protein 1 (PTBP1), whose nucleocytoplasmic translocation is involved in the rapid posttranscriptional up-regulation of insulin biosynthesis following islet stimulation with glucose and GLP-1. Glucose stimulated insulin secretion was decreased, albeit not significantly, in type 2 diabetic islets compared to non-diabetic islets. Stimulation increased the total amount of insulin (islet insulin content + secreted insulin) in islet preparation from non-diabetic patients, but not from type 2 diabetic subjects. Furthermore, the nuclear levels of PTBP1 were decreased in stimulated non-diabetic islets, but not in type 2 diabetic islets. These results suggest that impairment of rapid insulin increase in response to glucose is a specific trait of type 2 diabetic islets. Nuclear retention of PTBP1 is likely to play a role in this deficit, which in turn can contribute to impaired insulin secretion in type 2 diabetes. Overall, these data highlight the importance of investigating mechanisms of insulin biosynthesis and degradation to gain insight into the pathogenesis of type 2 diabetes.