Maize ( Zea mays ) is an important staple crop in many parts of the world but has low iron bioavailability, in part due to its high phytate content. Hemoglobin is a form of iron that is highly bioavailable, and its bioavailability is not inhibited by phytate. It was hypothesized that maize hemoglobin is a highly bioavailable iron source and that biofortification of maize with iron can be accomplished by overexpression of maize globin in the endosperm. Maize was transformed with a gene construct encoding a translational fusion of maize globin and green fluorescent protein under transcriptional control of the maize 27 kDa γ-zein promoter. Iron bioavailability of maize hemoglobin produced in Escherichia coli and of stably transformed seeds expressing the maize globin-GFP fusion was determined using an in vitro Caco-2 cell culture model. Maize flour fortified with maize hemoglobin was found to have iron bioavailability that is not significantly different from that of flour fortified with ferrous sulfate or bovine hemoglobin but is significantly higher than unfortified flour. Transformed maize grain expressing maize globin was found to have iron bioavailability similar to that of untransformed seeds. These results suggest that maize globin produced in E. coli may be an effective iron fortificant, but overexpressing maize globin in maize endosperm may require a different strategy to increase bioavailable iron content in maize.