ObjectiveThe free hormone hypothesis suggests that free and bioavailable 25-hydroxyvitamin D [25(OH)D] may better reflect vitamin D bioactivity. This study aimed to determine the free and bioavailable 25(OH)D characteristics, estimate their thresholds based on parathyroid hormone (PTH) and bone turnover markers (BTMs), assess their associations with the risk of metabolic syndrome (MetS), and evaluate their potential advantages.MethodsA cross-sectional study was conducted using a nationally representative database (n = 1,505, female, 18–45 years). Serum total 25(OH)D, vitamin D-binding protein, albumin, PTH, and BTMs [osteocalcin, β-CrossLaps of type 1 collagen containing cross-linked C-telopeptide (β-CTX), and procollagen type 1 N-terminal propeptide (P1NP)] were measured. Free 25(OH)D and bioavailable 25(OH)D were calculated. The threshold associations of 25(OH)D with PTH and BTMs were analyzed. The relationship between 25(OH)D and MetS risk was examined. An intervention study was then performed in 39 women (18–47 years) to assess the associations of increasing 25(OH)D with PTH and BTMs after vitamin D supplementation.ResultsIn the cross-sectional study, the three forms of 25(OH)D were found to have similar distribution characteristics. Free and bioavailable 25(OH)D correlated well with total 25(OH)D. Significant total 25(OH)D cutoffs were observed for PTH (14.19 ng/mL and 18.03 ng/mL), osteocalcin (15.14 ng/mL), β-CTX (14.79 ng/mL), and P1NP (15.08 ng/mL). Free and bioavailable 25(OH)D cutoffs were only found for P1NP (3.47 pg/mL and 1.66 ng/mL, respectively). A total 25(OH)D of