Technical Abstract: In the intestine, the epithelial cells are covered with a mucin layer produced by goblet cells. This mucin layer provides many important functions and can influence the uptake of iron. The objective of this study was to determine if a combination of commercially available mucin and an 8 um microporous membrane insert can be used to replace the 15 kD molecular weight cut-off (MWCO) dialysis membrane used in an established in vitro digestion/Caco-2 cell culture system. Although the current model with the 15 kD membrane correlates well with human studies, use of mucin may improve the system as the mucus layer is suspected to play a physiological role in Fe absorption. Use of mucin may also enable more complete assessment of iron bioavailability from large molecular weight forms of Fe such as heme and ferritin Fe. A range of foods or Fe (i.e. FeCl3 more/less than ascorbic acid (AA), cooked beef, red bean, white bean, soybean, horse spleen ferritin (HSF) and plant-type ferritin) were subjected to in vitro digestion. In the presence of mucin, significantly more Fe was taken up from the heme Fe (86%) and ferritin (91%) samples and significantly less Fe was taken up from the white bean samples (~70%) relative to the 15 kD membrane. The results indicated that the forms of iron interact with mucin. The mucus layer has a significant effect on Fe uptake. Further refinement and characterization of the mucin method is needed before it can be deemed a suitable replacement for the dialysis membrane method.