The immune system has the ability to distinguish between self and non-self. Also, Regulatory T-cells are a portion of Tcells that are in charge of preserving regulation of the immune system and immune tolerance through active immunerepression. Immune tolerance is very important to prevent the immune system from interacting against itself and thus to avoid the development of autoimmune diseasesInterleukin-35 (IL-35), which is a member of the interleukin-12 (IL12) cytokine family, is an anti-inflammatory immune response that plays the important function in the obstruction of the development of autoimmune diseases in different tests autoimmunities. When loss of self-tolerance occurs becuses many genetic and environmental factors, this causes the absence of balanced anti-inflammatory immune responses, which reduces the in vivo suppressive capacity of Treg, which is taken to be a regular T-cell-linked autoimmune disease., especially Hashimoto’s thyroiditis (HT). Methods: The current study included 90 samples. 60 hypothyroid HT patients (Group 1) and 30 healthy controls (Group 2) were enrolled in the study. Blood samples were collected and separated into serum from the two groups to be used to measure the functions of the thyroid gland by measuring the levels of thyroid hormone and thyroid stimulating hormone, measuring the level of anti-thyroid auto-antibodies, and determining the standard of serum Interloukin-35 through ELISA. Results: The results of the current study Through statistical analysis using the program SPSS version 22, it was shown that serum IL-35 levels were inversely Correlation used Pearson test with TSH, TPO-Ab, and TG-Ab in patients HT (-.292 (P≥0.01), -.245 (P≥0.02), -.269 (P≥0.01). While serum IL-35 levels were directly correlated with TSH, TPO-Ab, and TGAb in healthy controls (.151 (P≥0.01),.192 (P≥0.01),.097 (P≥0.01). The results of the current study showed a highly significant difference in TSH and FT4, respectively (P≥0.01), between the control and hypothyroid groups. Hypothyroidism is more common in females than males, and the incidence of hypothyroidism steadily increases with advancing age. Thyroid hormone concentrations show a significant change in patients with hormonal hypothyroidism compared with the control group. Conclusion: Our findings indicate an effective role for interleukin 35 in the reverse association with auto-antibodies and the development of autoimmune diseases, especially Hashmoto's disease