This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16–70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age ≤40 years) than in older donors and negatively correlated with the donor age (r = −0.535). Islet ATP was higher in younger donors (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/μg DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90–120 after clinical transplantation showed negative correlation with donor age (r = −0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in β-cells.