Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57Bl/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets.