Normal pregnancy is characterized by many changes in vitamin D metabolism, challenging the assessment of vitamin D status based exclusively on serum total 25-hydroxyvitamin D (25(OH)D). We hypothesized that measuring free and bioavailable fractions contributes to a better vitamin D status assessment in late pregnancy. Our aim was to evaluate a broad set of biomarkers of vitamin D status in Brazilian women in the third trimester of pregnancy. This cross-sectional study was conducted in women (n = 123, 18–44 y, 27–41 wk gestation) attended in a public maternity in Rio de Janeiro (2016–2018). Biomarkers included serum concentrations of total 25(OH)D3, parathyroid hormone (PTH), vitamin D-binding protein (DBP), and free and bioavailable fractions of 25(OH)D3. Vitamin D insufficiency (<50 nmol/L) was prevalent in 47.9% of the pregnant women. Serum 25(OH)D3 was inversely associated with the gestational week (β = −0.71, 95% confidence interval (CI): −1.31 to −0.16) and season, being lower in autumn (β = −9.90, 95% CI: −16.14 to -3.64) and winter (β = −16.74, 95%CI: −23.13 to −10.34). Concentrations of DBP, and free and bioavailable 25(OH)D3 were also inversely associated with winter months (P<0.05). DBP was directly associated with prepregnancy BMI (β = 5.84, 95% CI: 0.62 to 11.06). The recognized season-effect on total 25(OH)D3 appeared to also occur on free and bioavailable fractions. Although advanced gestational age was associated with lower total 25(OH)D3, our results suggest an adaptive mechanism responsible for maintaining free fraction during the 3rd trimester. We also suggest that starting pregnancy in obese condition may have an impact on vitamin D bioavailability, which deserves further investigation.