This review presents the applications of the in vitro cultures of human epithelial cells as a model for the study on functional properties of probiotic bacteria and bioactive food compounds. The main cell lines used in the research are Caco-2 as a model of small intestine and HT-29, as a model of large intestine. The structure, monolayer formation and physiology of epithelial cells are described. The commonly used growth media, culture vessels and growth conditions of the in vitro epithelial cultures are discussed. One of the most important applications of epithelial cell cultures are the studies on adhesion of probiotic bacteria and invasion of human pathogens. It was shown that Caco-2 and HT-29 cultures are suitable models for determination bacteria adherence. The mechanisms of adhesion and the role of environmental factors in bacteria attachment are discussed. The comparative studies on the bacteria adhesion to epithelial cells and solid substrates are described. The cell lines are also used in the studies on the trans-epithelial transport of different food compounds. In the review the results of experiments on the food compounds bioavailability are presented. Enterocyte-like cells from the in vitro cultures are also used to study carcinogenesis process caused by some chemical substances and free radicals from food products. The biological effects of the DNA damage on intestinal cell necrosis and apoptosis are discussed. Further research should confirm that epithelial cell cultures cultivated in laboratory conditions have qualities similar to those present under natural conditions in the human alimentary tract.