Heme-Fe is an important source of dietary iron in humans. Caco-2 cells have been used extensively to study human iron absorption, with emphasis on factors affecting nonheme iron absorption. We examined several factors known to affect heme-Fe absorption. Cells grown in bicameral chambers were incubated with high specific activity ⁵⁹Fe-heme alone or with 1% globin, bovine serum albumin (BSA), or fatty acid-free BSA (BSA-FA) to examine effect of protein source on absorption. Heme-Fe absorption was enhanced by globin and inhibited by BSA and BSA-FA. Absorption of heme-Fe in cells pre-treated for 7 days with serum-free medium containing 1, 25, 50, or 100 uM Fe was higher in the 1 uM Fe pre-treatment 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-Fe absorption and increased transport rate across the basolateral membrane. Finally, cells treated with 10 uM 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 makes this a promising model for examining intestinal heme-Fe absorption.