Abstract The divalent anion sodium symporter (DASS) family mediates the uptake of Krebs cycle intermediates and sulfate, and influences adiposity, insulin resistance, and metabolism in mammals. While Na+:substrate stoichiometry is known for several DASS transporters, the location of key Na+ binding sites remains elusive; important information for understanding the mechanism. In VcINDY, a bacterial DASS protein, we visualized a non-protein cryo-EM density in the middle of the transport domain. Its size and coordination suggest it may represent either a third Na+ ion or a structural water molecule. Using a combination of in vitro binding and transport assays, cryo-EM structural determination and molecular dynamic simulations, we show that the density is not a Na+ ion. Instead, the data indicate that the density likely represents a structural water molecule critical for transport domain integrity. Sequence and structural similarities suggest this feature may be conserved across human DASS transporters such as NaCT and NaDC3.