Although weight loss in older subjects has been shown to improve insulin sensitivity, it is unclear what effect this lifestyle intervention has on β-cell function. To determine whether diet-induced weight loss can improve β-cell function in older subjects, we studied 19 healthy male subjects (age, 65.4 � 0.9 yr; body mass index, 30.9 � 0.6 kg/m²; mean � sem) before and after a 3-month 1200-kcal/d diet. The insulin sensitivity index (S_I) was quantified using Bergman’s minimal model. The acute insulin response to glucose (AIRg) and the maximal glucose-potentiated insulin response (AIRmax) were determined and then adjusted for S_I (S_I � AIRg and S_I � AIRmax), thus providing measures of β-cell function. Subjects demonstrated significant weight loss (95.6 � 2.4 to 86.1 � 2.5 kg; P < 0.001). Both fasting plasma glucose [97.3 � 1.6 to 95.1 � 1.3 mg/dl (5.4 � 0.09 to 5.3 � 0.07 mm); P = 0.05] and insulin [18.5 � 1.3 to 12.2 � 1.0 μU/ml (110.9 � 7.7 to 73.5 � 5.9 pm); P < 0.001] levels decreased. With weight loss, S_I increased [1.59 � 0.24 to 2.49 � 0.32 � 10⁻⁴ min⁻¹/(μU/ml) (2.65 � 0.4 to 4.15 � 0.5 � 10⁻⁵ min⁻¹/pm); P < 0.001], whereas both AIRg [63.4 � 13.4 to 51.0 � 10.7 μU/ml (380 � 80 to 306 � 64 pm); P < 0.05] and AIRmax [314 � 31.4 to 259.9 � 33.4 μU/ml (1886 � 188 to 1560 � 200 pm); P < 0.05] decreased. Overall β-cell function improved (S_I � AIRg, 9.63 � 2.28 to 12.78 � 2.58 � 10⁻³ min⁻¹, P < 0.05; and S_I � AIRmax, 51.01 � 9.2 to 72.69 � 13.4 � 10⁻³ min⁻¹, P < 0.05). Thus, the weight loss-associated improvements in both insulin sensitivity and β-cell function may explain the beneficial effects of a lifestyle intervention on delaying the development of diabetes in older subjects.