Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria and into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in invasion but not in invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of but not of invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces invasion.