Breast cancer, as a significant threat to women’s health globally, has a complex pathogenesis. Vitamin D, a steroid hormone with diverse physiological functions, has garnered increasing attention in breast cancer research. Numerous studies have been conducted to elucidate the relationship between Vitamin D and breast cancer, yet no definitive conclusions have been reached. Hence, in this study, we conducted a two-sample Mendelian randomization (MR) analysis to investigate the association between Vitamin D and breast cancer. Genetic variants associated with Vitamin D levels and breast cancer data were obtained from the genome-wide association study database and version R11 of the FinnGen study dataset, respectively. The inverse variance-weighted (IVW) method served as the primary analytical approach, supplemented by various sensitivity analyses. Horizontal pleiotropy was tested using MR-Egger and MR-Pleiotropy RESidual Sum and Outlier methods, while sensitivity analysis was conducted using the leave-one-out method to assess the reliability of the results. Based on instrumental variable assumptions, 111 single nucleotide polymorphisms were suitable for subsequent analyses after matching the results. The MR analysis demonstrated no evidence of a causal relationship between Vitamin D and breast cancer. The IVW method yielded a non-significant association (p=0.968; odds ratio = 1.002, 95% confidence interval: 0.896 – 1.119), and the other methods pointed out the same direction of effect, and the subsequent pleiotropic analysis and sensitivity analysis confirmed the accuracy of the results. At the genetic level, no causal relationship between Vitamin D and breast cancer was found; thus, our findings do not support a clinically significant role of Vitamin D supplementation in breast cancer risk reduction.