Iron is one of essential micronutrient for all organisms. Its deficiency 33 causes a severe loss in crops yield. Nevertheless, our current understanding on major crops response to Fe deficiency remains limited. Herein, we investigated the effect of Fe deprivation at both transcriptomic and metabolic levels in hexaploid wheat. A genome-wide gene expression reprogramming was observed with a total of 5854 genes showing differential expression in roots of wheat subjected to Fe-starved medium. Subsequent, analysis revealed a predominance of strategy-II mode of Fe uptake, with induced genome bias contribution from the A and B genomes. In general, the predominance of genes encoding for nicotianamine synthase, yellow stripe like transporters, metal transporters, ABC transporters and zinc42 induced facilitator-like protein was noticed. Our transcriptomic data were in agreement with the GC-MS analysis that showed an enhancement of accumulation of various metabolites such as fumarate, malonate, succinate and xylofuranose, which could be linked for enhancing Fe-mobilization. Interestingly, Fe starvation causes a significant temporal increase of glutathione-S-transferase both at transcriptional and enzymatic activity, which indicate the important role of glutathione in the response to Fe starvation in wheat roots. Taken together, our result provides new insight on wheat response to Fe starvation and lays foundation to design strategies to improve Fe nutrition in crops.