OBJECTIVE: To evaluate the following from prepuberty to the puberty-onset: (1) changes in serum 25-hydroxyvitamin-D (25[OH]D), adiposity, and insulin resistance (IR); (2) the effect of prepubertal adiposity on serum 25(OH)D changes; and (3) the combined effect of prepubertal obesity and suboptimal-25(OH)D on IR at puberty-onset. METHODS: A total of 426 prepubertal children (∼54% girls) were followed during pubertal-onset assessing before and after puberty-onset serum 25(OH)D, adiposity (BMI and waist circumference) and IR indicators (homeostasis-model-assessment of IR [HOMA-IR]). Associations were tested using multiple and logistic regression models adjusted by age, gender, and seasonality. RESULTS: At puberty-onset, mean serum 25(OH)D decreased (32.2 ± 8.9 Tanner I vs 25.2 ± 8.3 ng/mL Tanner II) and total and central obesity increased (BMI-for-age-z-score ≥2 SD [%]: 16.4 vs 22.1; waist-circumference ≥75th percentile [%]: 27.2 vs 37.1, all P < .05). Children with higher adiposity before puberty onset had higher risk of suboptimal-25(OH)D (<30 ng/mL) in Tanner II (ie, odds ratio = 2.7 [1.1-6.7] for obesity and 2.7 [1.4-5.5] for central-obesity) after adjusting for relevant covariates. Children with higher adiposity and suboptimal-25(OH)D before puberty-onset had higher HOMA-IR compared with their counterparts in Tanner II (HOMA-IR: 2.8 [2.5-3.1] if central-obese and suboptimal-25[OH]D vs 2.1 [1.9-2.3] no central-obesity and optimal-25[OH]D). CONCLUSIONS: We found that serum 25(OH)D declined with puberty-onset, likely because of adiposity increase. Moreover, children with the combined condition of central-obesity and suboptimal-25(OH)D before puberty-onset had higher pubertal IR. These results highlight the need of ensuring adequate-25(OH)D status before pubertal-onset, particularly in obese children.