Heme-Fe is an important source of dietary iron in humans. Caco-2 cells have been used extensively to study human iron absorption with an emphasis on factors affecting nonheme iron absorption. Therefore, we examined several factors known to affect heme iron absorption. Cells grown in bicameral chambers were incubated with high specific activity [⁵⁹Fe]heme alone or with 1% globin, BSA, or fatty acid-free BSA (BSA-FA) to examine the effect of protein source on absorption. Heme iron absorption was enhanced by globin and inhibited by BSA and BSA-FA. Absorption of heme iron in cells pretreated for 7 days with serum-free medium containing 1, 25, 50, or 100 µM Fe was higher in the 1-µM-Fe pretreatment group than in all other groups (P < 0.05), showing an effect of iron status. Increased heme concentrations resulted in decreased percent absorbed but increased total heme iron absorption and increased transport rate across the basolateral membrane. Finally, cells treated with 10 µM CdCl₂, which induces heme oxygenase, demonstrated higher absorption of [⁵⁹Fe]heme than control cells (P < 0.05). Our results from Caco-2 cells are in agreement with human studies and make this a promising model for examining intestinal heme iron absorption.