Heme iron has been identified in many plant sourcesmost commonly in the root nodules of leguminous plants, such as soy. Our objective was to test the effectiveness of soy root nodule (SRN) and purified soy hemoglobin (LHb) in improving iron bioavailability using an in vitro Caco-2 cell model, with ferritin response as the bioavailability index. We assessed bioavailability of iron from LHb (either partially purified (LHbA) or purified (LHbD)) with and without food matrix and compared it with that from bovine hemoglobin (BHb), ferrous sulfate (FeSO4), or SRN. Bioavailability of each treatment was normalized to 100% of the FeSO4 treatment. When iron sources were tested alone (100 ug iron/mL), ferritin synthesis by LHbD and BHb were 19% (P > 0.05) and 113% (P < 0.001) higher than FeSO4, respectively. However, when iron sources were used for fortification of maize tortillas (50 ppm), LHbA and BHb showed similar bioavailability, being 27% (P < 0.05) and 33% (P < 0.05) higher than FeSO4. Heat treatment had no effect on heme iron but had a significant reduction on FeSO4 bioavailability. Adding heme (LHbA) iron with nonheme (FeSO4) had no enhancement on nonheme iron absorption. Our data suggest that heme iron from plant sources may be a novel value-added product that can provide highly bioavailable iron as a food fortificant.