Technical Abstract: We have generated transgenic maize plants expressing Aspergillus phytase either alone or in combination with the iron-binding protein ferritin. Our aim was to produce grains with increased amounts of bioavailable iron in the endosperm. Maize seeds expressing recombinant phytase showed enzymatic activities of up to 3 IU per gram of seed. In flour paste prepared from these seeds, up to 95% of the endogenous phytic acid was degraded, with a concomitant increase in the amount of available phosphate. In seeds expressing ferritin in addition to phytase, the total iron content was significantly increased. To evaluate the impact of the recombinant proteins on iron absorption in the human gut, we used an in vitro digestion/Caco-2 cell model. We found that phytase in the maize seeds was associated with increased cellular iron uptake, and that the rate of iron uptake correlated with the level of phytase expression regardless of the total iron content of the seeds. We also investigated iron bioavailability under more complex meal conditions by adding ascorbic acid, which promotes iron uptake, to all samples. This resulted in a further increase in iron absorption, but the effects of phytase and ascorbic acid were not additive. We conclude that the expression of recombinant ferritin and phytase could help to increase iron availability and enhance the absorption of iron, particularly in cereal-based diets that lack other nutritional components.