Background: Vitamin D deficiency is a global health problem and among Saudi subjects. However, vitamin D deficiency has been recognized to be involved in various immune functions, bone and muscle development. In addition, vitamin D deficiency has been shown to be associated with some autoimmune diseases. Only few reports have analyzed the supposed association between autoimmune thyroid diseases and vitamin D concentration with inconclusive results. Objective: This study was to examine the relationship, if any, between thyroid autoimmunity and Serum 25(OH) vitamin D levels in Saudi patients. Subjects and Methods: One hundred subjects were included in this study, which was carried out at outpatient endocrinology clinic of King Abdulaziz University Hospital (KAUH) in Jeddah, Kingdom of Saudi Arabia. Patients samples forwarded to immunology laboratory for thyroid antibodies, antithyroid peroxidase (TPO) and antithyroglobulin antibodies (Tg), analysis was selected and categorized as with high or normal levels of these antibodies and assayed for serum levels of 25(OH) vitamin D. Fifty five patients were found to have AITDs (evidenced by autoimmune features or elevated antithyroid peroxidase or antithyroglobulin antibodies) and forty five patients showed no evidence of autoimmunity (non-AITDs). Also, one hundred healthy subjects, age and sex-matched, volunteers living in the Jeddah area were randomly selected, as control group and anthropometric parameters, socioeconomic status were obtained after granted their consents. Results: Regards the serum 25(OH)vitamin D status, it was found to be more in the group (A) than in the group (B) (p = 0.024). In addition, the serum calcium, T3 and T4 level were found to be more in the group (A) than in the group (B) with statistical significant difference between both groups (p = 0.022, 0.023 and 0.019). While the serum TSH was found to be more in the group (B) than in the group (A) with statistical significant difference between both groups (p = 0.019). In addition, there were significant differences between AITD and non-AITD patients regarding serum 25(OH) vitamin D and abnormal thyroid function test. However, Pearson’s correlation coefficients test for the relationship between serum 25 (OH) vitamin D and each of serum antithyroid peroxidase (TPOAb) and antithyroglobulin (TgAb) antibodies levels in both groups showed a strong inverse relationship. Conclusion: There is a strong inverse relationship between serum 25 (OH) vitamin D and each of serum antithyroid peroxidase (TPOAb) and antithyroglobulin (TgAb) antibodies levels.