PURPOSE: Previous investigations have assessed the connection between vitamin D deficiency and an increased risk of gestational diabetes mellitus (GDM)
however, the findings remain inconsistent. The purpose of this study was to investigate the causal relationship between 25-hydroxyvitamin D (25OHD) levels and GDM. METHODS: Summary statistics data from genome-wide association studies (GWASs) were used to perform a bidirectional two-sample Mendelian randomization (MR) study. A total of 417,580 Europeans from the UK Biobank provided summary statistics data for 25OHD. The tenth data release of the FinnGen study provided the data for GDM, comprising 14,718 cases and 215,592 controls. For the univariate MR (uvMR) investigations, we employed the inverse variance weighted (IVW) method as our major analytical approach. Multiple sensitivity analyses were performed to evaluate the robustness of the results. Moreover, multivariate MR (mvMR) studies were conducted to account for potential confounding variables, including obesity, insulin resistance, and lipid traits. RESULTS: In the forward MR study, uvMR analysis did not provide evidence supporting a causal effect of 25OHD levels on the risk of GDM [IVW odds ratio (OR): 1.07, 95% confidence interval (CI): 0.95 to 1.19, p = 0.273]. After adjusting for obesity, fasting insulin levels, and lipid traits, the findings from the mvMR analysis aligned with those of the uvMR analysis. In the reverse MR study, uvMR analysis indicated that GDM had no causal effect on serum 25OHD levels (IVW β = -0.003, p = 0.804), and the robustness of this finding was confirmed in the mvMR study. CONCLUSION: Our MR research revealed no causal effect of serum 25OHD levels on GDM, suggesting that 25OHD deficiency does not correlate with an increased risk of GDM. Furthermore, our reverse analysis revealed no causal effect of GDM on 25OHD levels.