Nitrogen (N) is critical for micronutrient biofortification in wheat grain and is essential for a series of nitrogenous compounds biosynthesis. This study aims to assess the role of improved N supply in iron (Fe) and zinc (Zn) enrichment and expression of genes related to Zn and Fe chelation and transport in winter wheat. Potting and hydroponic culture experiments were conducted to study the effect of increasing N application on Zn and Fe uptake and translocation from roots to leaves and the temporal and spatial gene expression profiles of the NICOTIANAMINE SYNTHASE (NAS) genes in wheat. Plants were grown with low, medium and high N supply levels. The results showed that higher N application increased Fe and Zn content in leaves, and decreased Fe and Zn content in root compared with the lower N supply. High N application also increased the distribution of Fe and Zn from roots to leaves. Expression analysis showed that increased N application resulted in up-regulation of two wheat NAS genes, TaNAS1 and TaNAS2. Highly positive response between NAS genes and increasing N application indicated that abundance nicotianamine (NA) resulted from highly expressed NAS genes might involve in the chelation of Fe and Zn in the phloem and favor Fe and Zn uptake and accumulation in wheat leaves.