Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular iron deficiency anaemia is a major global health issue, but the iron content of staple crops such as wheat is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for biofortification of wheat (Triticum aestivum), we functionally characterized homologs of the Vacuolar Iron Transporter (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns, but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue growth of a yeast mutant lacking the vacuolar iron transporter. TaVIT2 also complemented a manganese transporter mutant, but not a vacuolar zinc transporter mutant. By over-expressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a > 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the UK. The anti-nutrient phytate was not increased and the iron in the white flour fraction was bioavailable in-vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and was also effective in barley. Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue bypassing existing homeostatic mechanisms.