The Na⁺/I⁻ symporter (NIS) mediates active I⁻ transport—the first step in thyroid hormonogenesis—with a 2Na⁺:1I⁻ stoichiometry. NIS-mediated ¹³¹I⁻ treatment of thyroid cancer post-thyroidectomy is the most effective targeted internal radiation cancer treatment available. Here to uncover mechanistic information on NIS, we use statistical thermodynamics to obtain K_ds and estimate the relative populations of the different NIS species during Na⁺/anion binding and transport. We show that, although the affinity of NIS for I⁻ is low (K_d=224 μM), it increases when Na⁺ is bound (K_d=22.4 μM). However, this K_d is still much higher than the submicromolar physiological I⁻ concentration. To overcome this, NIS takes advantage of the extracellular Na⁺ concentration and the pronounced increase in its own affinity for I⁻ and for the second Na⁺ elicited by binding of the first. Thus, at physiological Na⁺ concentrations, ~79% of NIS molecules are occupied by two Na⁺ ions and ready to bind and transport I⁻.