Available online xxxx Keywords: 25(OH)D3 Organochlorine compounds Persistent organic pollutants Polychlorinated biphenyls Pregnancy Vitamin D Persistent organic pollutants (POPs) are suggested to contribute to lower vitamin D levels; however, studies in humans are scarce and have never focused on pregnancy, a susceptibility period for vitamin D deficiency. We in-vestigated whether serum levels of POPs were associated with circulating 25-hydroxyvitamin D3 [25(OH)D3] concentration in pregnancy. Cross-sectional associations of serum concentrations of eight POPs with plasma 25(OH)D3 concentration were analyzed in 2031 pregnant women participating in the Spanish population-based cohort INfancia y Medio Ambiente (INMA) Project. Serum concentrations of POPs were measured by gas chromatography and plasma 25(OH)D3 concentration was measured by high-performance liquid chromatogra-phy in pregnancy (mean 13.3 ± 1.5 weeks of gestation). Multivariable regression models were performed to assess the relationship between blood concentrations of POPs and 25(OH)D3. An inverse linear relationship was found between serum concentration of PCB180 and circulating 25(OH)D3. Multivariate linear regression models showed higher PCB180 levels to be associated with lower 25(OH)D3 concentration: quartile Q4 vs. quar-tile Q1, coefficient = −1.59, 95% CI −3.27, 0.08, p trend = 0.060. A non-monotonic inverse relationship was found between the sum of predominant PCB congeners (PCB 180, 153 and 138) and 25(OH)D3 concentration: coefficient (95% CI) for quartile Q2 vs. Q1 [−0.50 (−1.94, 0.94)], quartile Q3 vs. Q1 [−1.56 (−3.11, −0.02)] and quartile Q4 vs. Q1 [−1.21 (−2.80, 0.38)], p trend = 0.081. No significant associations were found between circulating 25(OH)D3 and serum levels of p,p′-DDE, p,p′-DDT, HCB, and ß-HCH. Our results suggest that the back-ground exposure to PCBs may result in lower 25(OH)D3 concentration in pregnant women.