We have previously demonstrated that brain-derived neurotrophic factor (BDNF) ameliorates glucose metabolism and energy expenditure in obese diabetic db/db mice. In the present study, the effect of BDNF treatment on pancreatic islets of db/db mice was examined, using vehicle-treated pair-fed db/db mice as controls. Brain-derived neurotrophic factor (10 mg/kg) or vehicle was subcutaneously administered to male db/db mice for 4 weeks. The food intake of vehicle-treated db/db mice was restricted and precisely synchronized with that of BDNF-treated db/db mice using a pellet pair-feeding apparatus because BDNF decreases food intake in hyperphagic mice. Repetitive administration of BDNF significantly lowered the blood glucose concentration compared with pair-fed vehicle-treated db/db mice. The pancreatic insulin and glucagon concentrations were measured in db/db mice to evaluate the effect of BDNF on the pancreas. Although the insulin concentration in the pancreas of pair-fed vehicle-treated db/db mice was lower than in nondiabetic control +m/+m mice, it was higher in BDNF-treated db/db mice than in vehicle-treated pair-fed db/db mice and comparable to the concentration in +m/+m mice. The glucagon concentration in the pancreas of vehicle-treated pair-fed db/db mice was higher than in +m/+m mice, and BDNF partially decreased the glucagon concentration in the pancreas of db/db mice compared with vehicle. Histologic analyses of pancreatic sections were performed to characterize the mechanism through which BDNF modulates the hormonal concentration in the pancreas of db/db mice. Although there were no significant differences in the number and total area of islets between the BDNF- and vehicle-treated groups, immunostaining with an anti-insulin antibody indicated that the islet beta-cell area in BDNF-treated db/db mice was larger than that in vehicle-treated pair-fed db/db mice. Furthermore, immunostaining with an antiglucagon antibody indicated that BDNF normalized the delocalization of non-beta cells in islets of db/db mice. Electron microscopic images of beta cells indicated a decrease in secretory granules in vehicle-treated pair-fed db/db mice; this change was reversed in BDNF-treated db/db mice and reached a level comparable to that found in +m/+m mice. These findings suggest that BDNF prevents exhaustion of the pancreas in diabetic mice by maintaining the histologic cellular organization of beta cells and non-beta cells in pancreatic islets and restoring the level of insulin-secreting granules in beta cells.