Background: In this study, it was aimed to examine interleukin-35 (IL-35) levels and their association with thyroid function tests in Hashimoto’s thyroiditis (HT). Methods: Included in the study were 128 individuals, between 18 and 65 years of age, who had been newly diagnosed with HT (euthyroid, subclinical hypothyroidism and overt hypothyroidism) and 38 health controls who had no known diseases or drug use. Result: No significant difference was determined between the groups in terms of the IL-35 levels (P = 0.285), although in the in-group comparisons, the IL-35 levels were found to decrease progressively towards overt hypothyroidism. In the in-group comparisons, however, a statistically significant difference was determined between the control group and the overt hypothyroidism group (550.05 ± 411.50 vs. 369.80 ± 253.33; P = 0.046). When the patient groups were grouped according to their thyroid stimulating hormone values, a significant difference was determined between the groups with a threshold value of ?6 uIU/ mL and those below it, in terms of the IL-35 levels (P = 0.043). When two groups were created, comprising those with a threshold value of ?10 uIU/mL and those below it, it was observed that there was a more significant difference between the groups in terms of the IL-35 levels (P = 0.024). As a result of the correlation analysis performed by taking into account the controllable factors (smoking, diabetes mellitus, hypertension, and body mass index), a low-significant correlation was determined between the IL-35 levels and antithyroid peroxidase (P = 0.029). Conclusion: In conclusion, in this study, it was determined that the IL-35 levels, an antiinflammatory cytokine involved in HT, decreased progressively from the euthyroid patient group towards the overt hypothyroidism group.