Pancreatic islet transplantation is an effective treatment for diabetes mellitus, but it is not clear whether alpha-cell loss during islet isolation could impair the insulin release from beta-cells. To unravel this issue, human islets with alpha-cell deficiency were prepared by prolonged enzyme digestion, as confirmed by immunocytochemistry, immunofluorescence staining and islet insulin/glucagon content analysis. The functions of islets with alpha-cell deficiency were evaluated in vitro and in vivo. In vitro, human islets with alpha-cell deficiency exhibited low glucose-induced insulin release compared with intact islets. In islets deficient in alpha-cells, exogenous glucagon did not alone stimulate insulin release in the absence of glucose, but increased the glucose-induced insulin release in a dose-dependent manner. In intact islets, glucagon did not significantly change the glucose-stimulated insulin secretion. In vivo, transplantation of human islets with alpha-cell deficiency did not effectively correct hyperglycemia in diabetic C57BL/6 mice. In diabetic nude mice transplanted with islets deficient in alpha-cells, administration of exogenous glucagon significantly decreased glycemia, while withdrawing glucagon increased glycemic levels as compared with relevant controls. In addition, the survival of diabetic nude mice grafted with islets deficient in alpha-cells was significantly shorter than the survival of nude mice grafted with intact islets. These results indicated that glucagon-secreting alpha-cells have an important role in maintaining glucose-stimulated insulin release from beta-cells, and that alpha-cells loss from islets during isolation has a deleterious effects on insulin secretion.