Vitamin D insufficiency, as measured by 25-hydroxyvitamin D (25[OH]D) levels, has been associated with important health outcomes. The majority of vitamin D in circulation is bound to vitamin D–binding protein (DBP) and albumin, and recent genetic studies have demonstrated that serum DBP is a major determinant of 25(OH)D concentrations in adults. The impact of circulating DBP levels on vitamin D's biologic action, is unclear, but is of particular relevance to vitamin D epidemiology, because a lack of control for DBP levels could strongly influence the association of vitamin D with disease. Serum parathyroid hormone (PTH) levels can act as a biological readout of 25(OH)D activity. We therefore assessed the relationship between serum total and free 25(OH)D and PTH with and without adjusting for DBP, in 2073 subjects of European descent. Total 25(OH)D levels correlated positively (r = 0.19, p = 1.8 × 10−17) with DBP, whereas the free 25(OH)D correlated negatively (r = −0.14, p = 5.0 × 10−12). Total and free 25(OH)D levels correlated negatively with PTH (r = −0.29, p = 1.3 × 10−39; r = −0.26, p = 1.9 × 10−33, respectively). Including age, body mass index (BMI), sex, estimated glomerular filtration rate, calcium, and season of blood draw as covariates, total 25(OH)D levels were significantly associated with log-transformed PTH (lnPTH) levels (linear term: β = −0.010, p < 0.0001, squared term: β = 0.00004, p < 0.0001) and this association was not changed by adjusting for DBP. These findings provide evidence that in a largely vitamin D–sufficient cohort, the biological effect of vitamin D on PTH levels is mainly independent of DBP concentration. Accordingly, this study may provide useful information for studies investigating the relationship between vitamin D, DBP, and disease. © 2014 American Society for Bone and Mineral Research.