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Patulin (PAT) is a mycotoxin that grows on various fruits and grains, and its toxicity research mainly focuses on organ toxicity. However, the research on its impact on nutritional metabolism is still unclear. Vitamin D is an important nutrient and has been reported to have multiple physiological functions. Vitamin D deficiency has been reported to be associated with the risk of developing diseases such as fatal cancers, autoimmune disorders, cardiovascular disease, neurocognitive function impairment, and type 2 diabetes. However, vitamin D need to be converted into biologically active forms through two steps of hydroxylation in the liver or kidney by 25-hydroxylase (CYP2R1) and 25-hydroxyvitamin D 1-alpha-hydroxylase (CYP27B1). In this study, we found that PAT can cause liver and kidney injury, and affect the levels of activated vitamin D products 25(OH)D and 1,25(OH)₂D. Specifically, we demonstrated that PAT affects the hydroxylation of vitamin D by downregulating 25-hydroxylase (CYP2R1) and 25-hydroxyvitamin D 1-alpha-hydroxylase (CYP27B1) expression and interfering their substrate binding sites. This study can provide a basis for a more comprehensive assessment of the impact of environmental pollution, food safety, and other issues on human health.