Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H⁺-electrochemical potential gradient. The provenance of the H⁺ gradient in vivo is unknown so we have explored a role for brush-border Na⁺/H⁺ exchangers by examining iron homeostasis and intestinal iron handling in mice lacking Na⁺/H⁺ exchanger-2 (NHE2) or Na⁺/H⁺ exchanger-3 (NHE3). We observed modestly depleted liver iron stores in NHE2-null (NHE2^-/-) mice stressed on a low-iron diet but no change in hematological or blood-iron variables, or the expression of genes associated with iron metabolism, compared with wildtype mice. Ablation of NHE3 strongly depleted liver iron stores regardless of diet. We observed decreases in blood-iron variables but no overt anemia in NHE3^-/- mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase Cybrd1, and the basolateral iron exporter ferroportin was upregulated in NHE3^-/- mice, and expression of liver Hamp1 (hepcidin) suppressed, compared with wildtype. Absorption of ⁵⁹Fe from an oral dose was substantially impaired in NHE3^-/- mice compared with wildtype. Our data point to an important role for NHE3 in generating the H⁺ gradient that drives DMT1-mediated iron uptake at the intestinal brush border.