We studied the effects of hyperglycemia on β-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient β-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted β-cell mass (0.13 ± 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 ± 0.002 mg; group 4: 0.046 ± 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased β-cell apoptosis (group 1: 0.30 ± 0.05%; group 4: 0.42 ± 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 ± 0.09%; group 5: 0.48 ± 0.08%) compared with normal pancreas (0.04 ± 0.03%; P < 0.001). In contrast, on day 30, β-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 ± 0.03%) but not in normoglycemic mice (group 6: 0.12 ± 0.02%); β-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 ± 0.02 mg; group 6: 0.102 ± 0.009 mg; P < 0.01). In summary, even in optimal conditions, ∼60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to β-cell death. Increased apoptosis and reduced β-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted β-cells.