The sodium/iodide symporter (NIS) mediates active iodide (I⁻) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I⁻ transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I⁻. Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate (^99mTcO₄⁻). NIS KO mice showed undetectable serum T₄ and very low serum T₃ levels when fed a diet supplying the minimum I⁻ requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I⁻ diet partially rescued TH biosynthesis, demonstrating that, at high I⁻ concentrations, I⁻ enters the thyroid through routes other than NIS.