Introduction: Hashimoto thyroiditis (HT) is characterized by the presence of antithyroid antibodies with lymphocyte infiltration of the thyroid gland. Its etiology results from the interaction between environmental factors and genetic susceptibility traits. One of the factors most recently involved in autoimmunity is vitamin D. The principal aim of our study was to investigate a possible relationship between vitamin D status and antithyroid antibody levels in patients with newly diagnosed Hashimoto diseases, from Tlemcen. Materials and methods: Cross-sectional case-control study, including 310 subjects, divided into two groups: a group of cases (n = 155) with newly diagnosed HT and a group of apparently healthy controls (n = 155). The 02 groups were matched by sex, age ± 02 years, body mass index (BMI) and the same season (blood collection period). All subjects benefited from hormonal assays, anti-thyroid antibodies and biochemical parameters (Thyroid Stimulating Hormone (TSH), Free Thyroxine (FT4), anti-peroxydase antibody (anti-TPO), anti-thyroglobulin antibody (anti-Tg), 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), calcemia, phosphoremia, albuminemia and alkaline phosphatases). Results: High prevalence of hypovitaminosis D in our study population: 98% at the threshold of 25(OH)D < 30 ng/mL, 89% at the threshold < 20 ng/mL. The most significant risk factors associated with low levels of 25(OH)D are lack of sun exposure, season, skin color and lack of physical activity. The 25(OH)D deficit is more pronounced in the HT group than in the control group with a significant difference (11.45 ± 5.16 vs 13.02 ± 7.45; p = 0.03). In the HT group, the correlation between the status of 25(OH)D and the anti-TPO is inverse and significant (r = - 0.185; p = 0.02), relationship confirmed by multivariate analysis by linear regression where the 02 variables statistically related to anti-TPO are Vit D and TSH. Moreover, no correlation between 25(OH)D and anti-Tg and not between 25(OH)D and the degree of depth of biological hypothyroidism. Conclusion: There is an inverse and significant statistical relationship between serum levels of 25(OH)D and anti-TPO in the case group, however the exact involvement of vitamin D in the immunopathogenesis of Hashimoto’s diseases remains to be substantiated.