We measured the impact of chronic late gestation hypoglycemia on pancreatic islet structure and function in order to determine the cause of decreased insulin secretion in this sheep model of fetal nutrient deprivation. Late gestation hypoglycemia did not decrease pancreas weight, insulin content, -cell area, -cell mass, or islet size. The pancreatic islet isolation procedure selected a group of islets that were larger and had an increased proportion of -cells compared to islets measured in pancreatic sections, but there were no morphologic differences between islets isolated from control and hypoglycemic fetuses. The rates of glucose stimulated pancreatic islet glucose utilization (126.2±25.3 pmol glucose/islet/h, hypoglycemic vs. 93.5±5.5 pmol glucose/islet/h, control, p=0.47) and oxidation (10.5±1.7 pmol glucose/islet/h, hypoglycemic vs. 10.6±1.6 pmol glucose/islet/h, control) were not different in hypoglycemic fetuses compared to control fetuses. Chronic late gestation hypoglycemia decreased insulin secretion in isolated pancreatic islets by almost 70% in response to direct, non-nutrient membrane depolarization and in response to increased extracellular calcium entry. -cell ultrastructure was abnormal with markedly distended rough endoplasmic reticulum in three of the seven hypoglycemic fetuses studied, but in vitro analysis of hypoglycemic control islets showed no evidence that these changes represented endoplasmic reticulum stress, as measured by transcription of Glucose Regulatory Protein 78 and processing of X-box Binding Protein-1. In conclusion, these studies show that chronic hypoglycemia in late gestation decreases insulin secretion by inhibiting the later steps of stimulus-secretion coupling, after glucose metabolism, membrane depolarization, and calcium entry.