Introduction: Due to the high prevalence of vitamin D deficiency, strategies are needed to improve vitamin D status. Food components can affect vitamin D metabolism and have to be considered when estimating the efficacy of vitamin D supplements. 7-dehydrocholesterol (7-DHC) occurs naturally in food, but its impact on vitamin D metabolism has not yet been examined. Methods: Three groups of male C57BL/6 mice (n=12 per group) were placed on a diet that contained 0, 2.5 or 5mg 7-DHC per kg diet over a period of 6 weeks. Vitamin D and other sterols in the serum, skin, liver and kidney were quantified by LC-MS/MS. The relative mRNA abundance of hepatic genes encoding vitamin D hydroxylation enzymes and transporters was analyzed by real-time RT-PCR.Results: We found a substantial dose-dependent increase of non-hydroxylated vitamin D in the liver and kidney of mice fed a diet containing 7-DHC. The vitamin D content in the liver was 2.80±0.61pmol/g, 7.34±4.28pmol/g and 12.9±3.58pmol/g in groups that received 0, 2.5 and 5mg/kg 7-DHC, respectively. In the kidney, the vitamin D content of these groups was 1.78±1.17pmol/g, 3.55±1.06 and 6.36±2.29pmol/g, respectively. The serum and tissue concentrations of 25-hydroxyvitamin D (25(OH)D) remained unaffected by 7-DHC. The relative mRNA data provided no plausible mechanism for the observed effects of 7-DHC on vitamin D. All groups of mice had similar concentrations of cholesterol, desmosterol and 7-DHC in their serum and tissues.Conclusion: The current findings provide the first evidence that dietary 7-DHC seems to affect vitamin D metabolism. The underlying mechanism remains elusive and needs further investigation.