The aim of the presented work was the functional comparison of conventional 21-day-old and modified 6-day-old Caco-2 cell cultures for their application in studies on the bacterial adhesion and transepithelial transport. In the short-term culture system, the cells were grown on polycarbonate filters in serum reduced medium supplemented with a combination of growth factors. Morphological comparison of two Caco-2 monolayers showed that the short-term culture had the similar ultrastructural characteristics of the long-term well-developed culture. The transepithelial electrical resistance measurement confirmed the high Caco-2 monolayer integrity in both cell culture systems. As a result, the modified 6-day-old Caco-2 culture could be applied in permeability and adhesion investigations. In the transport study, atrazine was used as a model molecule. The transport of atrazine was symmetric between the apical to basolateral and the basolateral to apical directions and independent of its concentration. No significant difference was found in atrazine permeability through conventional and modified Caco-2 cultures. In order to investigate the role of Caco-2 culture protocol might play in the adhesion of probiotic bacteria, three lactobacilli strains were used. The effect of Caco-2 culture modification on adhesion capacity of all tested bacteria was not observed. The proposed system of Caco-2 cell culture can be a considerable alternative to the conventional 21-day system.