Haemolytic anaemia induced by phenylhy‐drazine (PZ) promotes iron absorption across rat small intestine. This present study investigates the role of the brush border potential difference (V_m) and mucosal reducing activity in the response. In addition, quantitative autoradiography was used to assess PZ‐induced changes in the villus localization of brush border iron uptake. Iron transfer from duodenum to blood was increased significantly 5 days after treatment with PZ. Autoradiography showed that most brush border iron uptake occurred at the upper villus region and the maximal rate was increased fourfold by PZ. Duodenal villus length was increased in PZ‐treated rats. PZ treatment did not influence mucosal reducing activity but V_m, measured using duodenal sheets, increased from ‐50 to ‐57mV {P < 0.001) and this was due to a reduced brush border sodium permeability. Thus, an expanded absorptive surface and an enhanced electrical driving force for iron uptake across the duodenal brush border are important adaptations for increased iron absorption in PZ‐induced haemolytic anaemia.