INTRODUCTION: Numerous physiological systems, such as the functioning of the immune system, bone health, and the regulation of expression of genes, depend critically on vitamin D. Considering the significance of vitamin D for health, it is critical to understand how it is metabolized and the factors that affect its levels. METHODS: The objective of this study was to develop and validate an LC-MS/MS method to examine the effects of light exposure and dietary vitamin D consumption on the levels of vitamin D and its metabolites in a mouse model under consistent growth conditions throughout the year. Serum and hair samples from mice were analyzed under various experimental conditions for vitamin D and its metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experimental conditions included a vitamin D-deficient diet, a vitamin D-standard diet, and changes in ambient light exposure ranging from complete darkness to a regular light-dark cycle. RESULTS: Mice fed a standard vitamin D diet and exposed to a regular light-dark cycle exhibited significantly higher levels of 25OHD DISCUSSION: This study provides valuable insights into the complex interplay between dietary vitamin D intake, light exposure, and the regulation of vitamin D metabolism in mice. Moreover, our results underscore the potential implications for human health, suggesting the importance of adequate vitamin D intake and sunlight exposure in maintaining optimal vitamin D levels. Further research in this area has the potential to unveil additional factors influencing vitamin D metabolism, offering valuable insights into strategies for optimizing vitamin D levels in both animal models and human subjects.