Previous cross-sectional studies show that puberty is associated with a reduction in insulin sensitivity (SI), but no longitudinal studies have examined this change in detail. This study is a longitudinal study in 60 children (33 male and 27 female subjects; 32 Caucasian and 28 African-American) examined at Tanner stage I (age 9.2 ± 1.4 years) and after 2.0 ± 0.6 years of follow-up, by which time 29 children remained at Tanner stage I and 31 had progressed to Tanner stage III or IV. Tanner stage was assessed by physical examination. SI, the acute insulin response (AIR), and the disposition index (DI) were determined by the tolbutamide-modified intravenous glucose tolerance test and minimal modeling, body fat mass was assessed by dual-energy X-ray absorptiometry, visceral fat was determined by computed tomography, and fasting blood was analyzed for hormone levels. In children progressing to Tanner stage III, SI fell significantly by 32% (4.4 ± 3.0 to 3.0 ± 1.7 × 10-4min-1[μIU/ml]), AIR increased by 30%, DI fell by 27%, and there was a significant increase in fasting glucose (93.5 ± 5.0 to 97.0 ± 4.1 mg/dl) and insulin (14.3 ± 8.1 to 18.6 ± 11.0 μIU/ml). In children remaining at Tanner stage I, there was a slight increase in SI (6.4 ± 3.1 to 7.4 ± 3.5 × 10-4min-1[μIU/ml]) with no significant change in AIR or fasting glucose and insulin. The pubertal fall in SI was more consistent in African-Americans; remained significant after controlling for age, sex, and change in fat mass, visceral fat, and fat-free mass; and was similar in children at low, medium, and high body fat. Change in SI was not significantly related to change in fasting hormone levels, but change in AIR was significantly related to change in androstendione (r = 0.39; P = 0.04). Pubertal transition from Tanner stage I to Tanner stage III was associated with a 32% reduction in SI and increases in fasting glucose, insulin, and AIR. These changes were similar across sex, ethnicity, and obesity. The significant fall in DI suggests conservation in β-cell function or an inadequate β-cell response to the fall in SI. The fall in SI was not associated with changes in body fat, visceral fat, IGF-I, androgens, or estradiol.