Evidence on the effect of ambient air pollution on vitamin D is limited. This study aimed to examine the association of air pollution exposure with serum levels of 25-hydroxyvitamin D (25OHD) using UK Biobank health datasets. A total of 448,337 subjects were included in this analysis. Land Use Regression was applied to assess individual exposures to particulate matter with diameters ≤2.5 µm (PM 2.5), ≤10 µm (PM 10), nitrogen dioxide (NO 2), and nitrogen oxides (NO x). Linear regression models evaluated the associations between air pollutants and serum vitamin D levels after adjustment of a series of confounders. All analyzed air pollutants were negatively associated with serum vitamin 25OHD levels. After adjusting for potential confounders, a 10 μg/m 3 increase in concentrations of PM 2.5, PM 10, NO x, and NO 2 was associated with −9.11 (95%CI: −13.25 to −4.97), −2.47 (95%CI: −4.51 to −0.43), −0.56 (95%CI: −0.82 to −0.30), and −1.64 (95%CI: −2.17 to −1.10) nmol/L decrease in serum vitamin 25OHD levels, respectively. Interaction analyses suggested that the effects of air pollution were more pronounced in females. In conclusion, long-term exposures to ambient PM 2.5, PM 10, NO x, and NO 2 were associated with vitamin D status in a large UK cohort.