Previously, we have demonstrated increased iron absorption from low molecular weight (LMW) human milk whey fractions. In the present study, we investigated the effect of heat denaturation, zinc (a competitor of iron), duodenal cytochrome b (DcytB) antibody neutralization and citrate lyase treatment on LMW human milk fraction (>5 kDa referred as 5kF) induced ferric iron reduction, solubilization, and uptake in Caco-2 cells. Heat denaturation and zinc inhibited the 5kF fraction induced ferric iron reduction. In contrast, zinc but not heat denaturation abrogated the ferric iron solubilization activity. Despite inhibition of ferric iron reduction, iron uptake in Caco-2 cells was similar from both native and heat denatured 5kF fractions. However, iron uptake was higher from native compared to heat denatured 5kF fractions in the cells preincubated with the DcytB antibody. Citrate lyase treatment inhibited the ferric iron reduction, solubilization, and uptake in Caco-2 cells. These findings demonstrate that citric acid present in human milk solubilizes the ferric iron which could be reduced by other heat labile components leading to increased uptake in intestinal cells.