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. (J Clin Endocrinol Metab 99: 178-183, 2014). © Copyright 2014 by The Endocrine Society.