CONTEXT: We hypothesized that, similar to the coordinated homeostatic regulation of most hormones, the concentration of free and bioavailable 25-hydroxyvitamin D [25(OH)D] will be tightly controlled by total 25(OH)D and vitamin D binding protein (VDBP) and that the VDBP concentrations will be associated with insulin resistance status. OBJECTIVE: Our primary objective was to investigate associations between total, free, and bioavailable 25(OH)D and VDBP. We also evaluated the relationships of VDBP with insulin resistance indices. STUDY DESIGN: The study design was cross-sectional in the setting of a university children's hospital. The relative concentration of bioavailable 25(OH)D to total 25(OH)D [bioavailable 25(OH)D/total 25(OH)D was expressed as a percentage [percentage bioavailable 25(OH)D]. RESULTS: Subjects were 47, postmenarchal, female adolescents, with a mean age of 15.8±1.4 years, a mean body mass index of 23.1±4.0 kg/m2. The total 25(OH)D was strongly associated with VDBP (rho=0.57, P<.0001). At lower total 25(OH)D concentrations, the concentration of bioavailable 25(OH)D relative to total 25(OH)D was higher (23.8% vs 14.9%, P<.0001), whereas the relative concentration of free 25(OH)D was similar (P=.44). VDBP was inversely associated with fasting insulin (rho=-0.51, P=.0003) and homeostatic model assessment of basal insulin resistance (rho=-0.45, P=.002) and positively with whole-body insulin sensitivity (rho=0.33, P=.02); these relationships persisted after adjusting for percentage fat and attenuated after adjusting for race. CONCLUSION: Our data suggest that VDBP concentrations are regulated by total 25(OH)D levels to maintain adequate concentrations of bioavailable 25(OH)D. VDBP concentrations are inversely associated with hyperinsulinemia and insulin resistance.