The Caco-2 cell model is widely used to assess the bioaccessibility/availability of iron from foods and diets. Analysis of iron uptake in this human epithelial cell line is usually preceded by a two-step digestion to simulate the conditions in the stomach and small intestine. Moreover, culturing the cells on inserts permits the measurement of iron transport. The cellular iron uptake is determined by direct measurements using radioisotopes, or indirectly by measurement of ferritin, the intracellular storage form of iron. There is a good correlation between Caco-2 cell uptake and human iron bioavailability for a number of dietary factors known to affect iron absorption. However, recent data suggest that in some cases there is no correlation. Possible reasons for such discrepancies, the benefits, and limitations of the Caco-2 cell model are discussed. In conclusion, in vitro experiments with Caco-2 cells are important tools for ranking foods with respect to bioavailability, for mechanistic studies of iron absorption, and for studies of dietary factors influencing absorption. The results need to be confirmed in humans.