Objective To evaluate the relationship between Hashimoto’s thyroiditis (HT) and 25-hydroxy vitamin D. Methods A total of 334 HT patients were enrolled from the endocrine department of our hospital from January 2017 to December 2019, and 300 non-HT patients were selected as the control. There was no significant difference in gender and age composition between the two groups(P>0.05). The indexes of liver function, renal function, blood glucose, blood lipid, electrolyte level, thyroid function, thyroid antibody and vitamin D level of the two groups were collected and analyzed. Results Compared with the control group, there was no significant difference in vitamin D level and vitamin D deficiency rate between the HT group and the control group (P>0.05), while the thyroid stimulating hormone (TSH) level and the incidence of subclinical hypothyroidism in the HT group was significantly higher than that in the control group(P<0.05). The vitamin D level of female HT patients was lower and the cases of subclinical hypothyroidism were more(P<0.05). The HT patients with vitamin D deficiency were more likely to show subclinical hypothyroidism than the HT patients with normal vitamin D. Serum free triiodothyronine(FT₃) was a protective factor for 25-hydroxy vitamin D[odds ratio(OR)=0.958, P=0.002], serum TSH(OR=1.080, P=0.002) and female(OR=1.167, P=0.001) were risk factors for 25-hydroxy vitamin D. Conclusions The vitamin D deficiency is prevalent in the population, especially in female. Vitamin D deficiency can lead to higher TSH level and increase the incidence of subclinical hypothyroidism in HT patients.