Low serum 25-hydroxyvitamin D3 (25(OH)D) levels have been associated with insulin resistance and cardiovascular diseases. The influences of gender, puberty and adiposity on vitamin D status and the relationship between 25(OH)D and cardiometabolic risk factors in obese and non-obese children were studied. A retrospective analysis was carried out on 168 Turkish children during late winter. Age, gender, puberty, body mass index (BMI), 25(OH)D levels and cardiometabolic risk factors including lipid profiles, high-sensitivity C-reactive protein and insulin resistance index calculated by homeostasis model assessment (HOMA-IR) were evaluated. The median age of the study population was 11 (4–16) years, and 102 children (60.7 %) were prepubertal. Overall, 98.2 % of patients had 25(OH)D levels lower than 20 ng/mL (median 10.0 (4.0–21.3) ng/mL). The 25(OH)D levels did not correlate with BMI. However, an inverse correlation was seen between serum 25(OH)D and HOMA-IR (rho = −0.656, p = 0.006) and insulin (rho = −0.715, p = 0.002) in pubertal obese subjects. Female gender and puberty were all negatively associated with 25(OH)D. Conclusion: The association between vitamin D status and BMI is complex, and it does not seem to be altered by mild obesity. In addition, potential influence of puberty should be kept in mind while assessing the relationship between serum 25(OH)D and cardiometabolic risk factors.