The invasive properties of cancer cells depend on their intrinsic motile potential and on their ability to breach the endothelial barrier. In the present work, we investigated the mechanisms by which adhesion of colon cancer cells to E-selectin expressed by endothelial cells regulates the barrier function of these cells and modulates transmigration of cancer cells. We found that the stimulation of E-selectin by activating antibodies or the adhesion of HT-29 cells results in an increase in the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases. In turn, the activation of p38 and ERK enhances transendothelial permeability and migration of HT-29 cells. We also obtained evidence suggesting that p38-mediated increase in transendothelial migration of cancer cells depends on a myosin light chain phosphorylation-mediated formation of stress fibres. On the other hand, the activation of ERK by E-selectin modulates the opening of interendothelial spaces by initiating the activation of Src kinase activities and the dissociation of the VE-cadherin/beta-catenin complex. We conclude that activation of E-selectin by adhering cancer cells is an important process that regulates the extravasation of colon cancer cells by initiating p38- and ERK-dependent mechanisms that both contribute to regulate the integrity of the endothelial layer.