Root release of phytosiderophores (PSs) is an important step in iron (Fe) acquisition of grasses, and this adaptive reaction of plants is affected by various plant and environmental factors. The objectives of this study were to study the effects of varied nitrogen (N) supply on (1) root and leaf concentrations of methionine, a precursor in the PS biosynthesis, (2) PS release from roots, (3) mobilization and uptake of Fe from 59Fe-labeled Fe(III)-hydroxide [59Fe(OH)3] and (4) root uptake of 59Fe-labeled Fe(III)–deoxymugineic acid (DMA) by durum wheat (Triticum durum, cv. Balcali2000) plants grown in a nutrient solution. Enhanced N supply from 0.5 to 6 mM in a nutrient solution significantly increased the root release of PS under Fe deficiency. High N supply was also highly effective in increasing mobilization and root uptake of Fe from 59Fe-hydroxide under low Fe supply. With adequate Fe, N nutrition did not affect mobilization and uptake of Fe from 59Fe(OH)3. Root uptake and shoot translocation of Fe supplied as 59Fe(III)–DMA were also stimulated by increasing N supply. Leaf concentration of methionine was reduced by low Fe supply, and this decline was pronounced in high N plants. The results show that the root release of PS, mobilization of Fe from 59Fe(OH)3 and root uptake and shoot translocation of Fe(III)–PS by durum wheat are markedly affected by N nutritional status of plants. These positive N effects may have important implications for Fe nutrition of human populations and should be considered in biofortification of food crops with Fe.