The human colon adenocarcinoma cell line HT29, is not only used to study the biology of human colon cancers, but it is receiving special interest in studies focused on food digestion and bioavailability due to the ability to express characteristics of mature intestinal cells. In the differentiated phenotype, they are able to form a monolayer with tight junctions between cells and a typical apical brush border. In addition, these differentiated cells express brush-border-associated hydrolases typical of the small intestine although the enzymatic activity is lower than that found in vivo. Although they represent a valuable model due to their similarities with enterocytes of the small intestine, their limitations and the relevance to the in vivo situation are also considered in this chapter. The application of this cell line to transport studies of drugs and food compounds is illustrated, especially when the effect of the mucus layer is considered or used as co-culture in combination with Caco-2 cells. They have also been frequently used to study the intestinal immune response to bacterial infection, and microorganism survival, adhesion or invasion. Finally, the use of these cells to evaluate the effect of several food compounds and mucin secretion is summarized.