Significance The Na + /I − symporter (NIS) uses the Na + electrochemical gradient to actively transport I − into the thyroid with a 2 Na + :1 I − stoichiometry. In NIS and related transporters, one of the two Na + binding sites, Na2, includes a Ser and a Thr residue. We determined that, besides these residues, four other amino acids participate in binding Na + at the Na2 site, at different stages of the transport cycle. These residues are conserved throughout the entirety of solute carrier family 5 (SLC5), to which NIS belongs. Replacing any of these residues has a marked effect on transport and on the dynamics of NIS. These results not only deepen our understanding of the mechanism of transport by NIS but are also relevant to the study of other Na + -driven cotransporters.