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Purpose: The association between autoimmune thyroid diseases (AITDs) and vitaminD deficiency is controversial. We aimed to evaluate the relationship between serum25-hydroxy-vitamin D3 [25(OH)D3] and anti-thyroid antibody levels. Materials and Methods: 25(OH)D3, anti-thyroid antibodies, and thyroid function measured in 304 patients who visited the endocrinology clinic were analyzed. The patients were subgrouped into the AITDs or non-AITDs category according to the presence or absenceof anti-thyroid antibodies. The relationship between anti-thyroid peroxidase antibody (TPOAb) and 25(OH)D3 was evaluated. Results: The patients with elevatedanti-thyroid antibodies had lower levels of serum 25(OH)D3 than those who did not (12.6±5.5 ng/mL vs. 14.5±7.3 ng/mL, respectively, p<0.001). Importantly, after adjusting for age, sex, and body mass index, a negative correlation (r=-0.252, p<0.001) was recognized between 25(OH)D3 and TPOAb levels in the AITDs group, but this correlation did not exist in the non-AITDs group (r=0.117, p=0.127). 25(OH)D3 level was confirmed as an independent factor after adjusting for co-factorsthat may affect the presence of TPOAb in the AITDs group. Conclusion: 25(OH)D3 level is an independent factor affecting the presence of TPOAb in AITDs. The causal effect of 25(OH)D3 deficiency to AITDs is to be elucidated.