Background Despite numerous observational studies on the association between serum 25-Hydroxyvitamin D levels and cutaneous melanoma, causal inferences remain ambiguous due to confounding and reverse causality. This study aimed to elucidate the causal relationship between serum 25-Hydroxyvitamin D levels and melanoma incidence using Mendelian randomization (MR). Methods A two-sample MR was conducted using genetic variants associated with serum 25-Hydroxyvitamin D levels as instrumental variables. Summary statistics for these variants were derived from genome-wide association studies, and those for melanoma risk were obtained from a comprehensive melanoma case-control study. Robustness of the results was assessed through sensitivity analyses, including the “leave-one-out” approach and tests for potential pleiotropy. Results The MR analysis provided substantial evidence of a positive causal relationship between serum 25-Hydroxyvitamin D levels and the incidence of cutaneous melanoma, suggesting that each unit increase in serum 25-Hydroxyvitamin D levels corresponds with an increased risk of melanoma. Tests for pleiotropy showed minimal effects, and the sensitivity analysis confirmed no disproportionate influence by any individual single nucleotide polymorphism (SNP). Conclusion The findings indicated a potentially causal positive association between serum 25-Hydroxyvitamin D levels and melanoma risk, challenging traditional beliefs about vitamin D’s role in melanoma. This emphasizes the need for a balanced and personalized approach to vitamin D supplementation and sun exposure, particularly in high-risk populations. These results should be interpreted with caution due to potential unrecognized pleiotropy and confounding factors. Future research should focus on validating these findings in diverse populations and exploring underlying biological mechanisms.