Background: Nitric oxide (NO) plays a key role in thyroid function regulation through the inhibition of iodide (I) uptake at the thyroidal sodium-iodide symporter (NIS) and impacts the thyroid vascularity and blood flow. Objectives: This study aimed to evaluate the association between serum NO metabolites (NOx) and thyroid-stimulating hormone (TSH), free thyroxin (FT4), and anti-thyroid peroxidase (TPOAb) changes. Also, it aimed at evaluating the correlation between serum NOx and the incidence of clinical hypothyroidism, characterized by elevated TSH level and decreased FT4 concentration, and subclinical hypothyroidism, characterized by mildly elevated TSH level despite FT4 concentration within the normal range, over three years of follow-up. Methods: This study included 1,137 participants of the Tehran Thyroid study (TTS), aged > 20 years old, for whom data on serum TSH, FT4, and TPOAb in the third and fourth phases, and serum NOx in the third phase were available. Changes in TSH (ΔTSH), FT4 (ΔFT4), and TPOAb (ΔTPO) between the third and fourth phases were calculated, and the associations between serum NOx and ΔTSH, ΔFT4, and ΔTPOAb were assessed after multivariable adjustment using linear regression analysis. Results: No significant association was found between serum NOx and ΔTSH, ΔFT4, and ΔTPOAb after the multivariable adjustment; neither was any observed in TPOAb split groups after multivariable adjustment. No significant association was found between serum NOx tertiles and clinical and subclinical hypothyroidism incidence in the fourth phase of TTS. Conclusions: There was no association between serum NOx levels and changes in TSH, FT4, and TPOAb and clinical and subclinical hypothyroidism incidence.