Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an iron-enriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate.