Abstract: Recent studies have revealed an association between nitrogen oxide (NO_x) concentrations and 25-hydroxyvitamin D (25(OH)D) concentrations; however, the causal relationship between them remains unclear. To investigate this further, we utilized data from the British Biobank (MRC-IEU) with the IEU Open GWAS database. Genetic loci that were independently and strongly associated with nitrogen oxides were selected as instrumental variables (IVs). The inverse variance weighting method (IVW) was employed as the primary approach, complemented by a weighted median model, MR-Egger regression, a simple model, and a weighted model within a two-sample Mendelian randomization (TSMR) framework. The odds ratio (OR) was used as an evaluation index to study their causal relationship. The results showed that: (1) After rigorous screening and evaluation, we selected seven nucleotide polymorphisms (SNPs) as instrumental variables. The IVW method indicated a potential causal relationship between nitrogen oxide exposure and 25-hydroxyvitamin D concentrations (P<0.01), with an odds ratio (OR) of 0.74 (95% CI: 0.62-0.87). This further confirms a negative correlation between nitrogen oxide exposure and 25-hydroxyvitamin D concentrations. (2) A comprehensive examination of the results of the Mendelian randomization analysis included multiple testing, heterogeneity testing, and stepwise exclusion testing. The Mendelian randomization Egger method was used to evaluate the multivariate effects and heterogeneity of the instrumental variable. The results of the multiple testing showed that the intercept term was -0.0041 (P=0.53), and the heterogeneity test yielded a P value of 0.07. After excluding one instrumental variable, the estimated Mendelian randomization results for the remaining instrumental variables were not significantly different from the overall results. These findings indicate that the negative correlation between nitrogen oxides and 25-hydroxyvitamin D concentration is robust. Overall, these results suggest that nitrogen oxide exposure may increase the risk of decreased serum 25-hydroxyvitamin D levels in European populations. Future research should focus on exploring the specific mechanisms underlying this relationship and assessing its potential long-term impact on human health.