Objective To study the protective mechanism of 1, 25 (OH)₂D₃ in experimental autoimmune thyroiditis rats based on the TLR2 / NF-κB signaling pathway. Methods Rats were randomly divided into control, model, and selenium yeast tablet control groups, and 1,25(OH)₂D₃ low, medium and high dose groups. Except for the control group, the rats were injected subcutaneously with thyroglobulin (PTg) to establish an autoimmune thyroiditis model. The low, medium and high dose groups of 1,25(OH)₂D₃ were injected intraperitoneally with 0. 5, 1. 0 and 1. 5 μg / kg 1,25 (OH)₂D₃ , the control and model groups were injected with the same amount of distilled water and selenium yeast tablets, and the control group was injected with the same amount of selenium yeast suspension ( once a day) for 4 weeks. Thyroid function factors, serum inflammatory factors, thyroid autoantibodies, TLR2 / NF-κB signaling pathway-related protein levels, and gene expression were analyzed. Results Compared with autoimmune thyroiditis rats, serum free thyroid glycine, free thyroxine, thyroid-stimulating hormone, thyroglobulin antibody, and thyroid peroxidase antibody, and interleukin (IL)-6, IL-12, tumor necrosis factor α (TNF-α), thyroid tissue TLR2, MyD88, TRAF-6 and NF-κB mRNA and protein expression were reduced, and IL-10 was increased in 1,25(OH)₂D₃ group rats. The difference between high dose 1,25(OH)₂D₃ and model groups was statistically significant (P< 0.05). Conclusions 1,25(OH)₂D₃ improves the thyroid functions of rats with autoimmune thyroiditis and increases the level of autoimmune antibodies. The mechanism may be related to inhibition of the TLR2 / NF-κB signaling pathway by 1, 25 (OH)₂D₃ , which regulates the release of inflammatory factors such as IL-6, IL-10, IL-12 and TNF-α.