Background: serum 25-hydroxyvitamin D (25(OH)D) (“total 25 OH(D)”) is the most commonly used indicator of vitamin D status. However, 25(OH)D is mostly bound to the vitamin D binding protein (VDBP) or albumin in blood, and it has been suggested that the remaining bioavailable or free 25(OH)D may be more relevant for vitamin D associated health outcomes. We aimed to explore distributions and determinants of VDBP, total, bioavailable, complementary “non-bioavailable”, and free 25(OH)D in a large cohort of older adults in Germany. Methods: total 25(OH)D, VDBP, and albumin concentrations were measured in blood samples of 5899 men and women aged 50–75 years and used to calculate bioavailable (and complementary “non-bioavailable”) and free 25(OH)D concentrations. Linear regression models were used to evaluate associations of potential determinants of the various vitamin D biomarkers. Results: mean concentrations of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D were 323.6 µg/mL, 49.8 nmol/L, 43.4 nmol/L, 2.5 ng/mL, and 5.7 pg/mL, respectively. Seasonal variations were observed for all markers, with peak values in spring for VDBP and in summer for total, non-bioavailable, bioavailable, and free 25(OH)D. Consistent inverse associations were seen with age and body mass index for all markers, but divergent associations were seen with C-reactive protein. Strong variations by VDBP genotypes were seen for bioavailable and free 25(OH)D, and, in opposite direction for non-bioavailable 25(OH)D. Conclusion: commonalities and differences in determinants of various markers of vitamin D status were observed, which may help to enable a better understanding of their potential role for various vitamin D related health outcomes.