Invasion is an important microbial virulence strategy to overcome the barrier formed by polarized epithelial cells. Salmonella enterica is a food-borne pathogen that deploys a type III secretion system for the manipulation of the actin cytoskeleton and to trigger internalization into epithelial cells. Here we show that this function is not sufficient to enter polarized cells and report that penetration of epithelia from the luminal side requires both the type III secretion system and novel virulence functions conferred by Salmonella pathogenicity island 4. Salmonella pathogenicity island 4 encodes a type I secretion system for the giant non-fimbrial adhesin SiiE that mediates intimate contact of Salmonella to microvilli on the apical membrane. Mutant strains lacking SiiE fail to invade polarized cells, to destroy epithelial barrier functions and to efface the apical brush border. Deletion analyses of repetitive domains in SiiE indicate that the large size of the adhesin is of functional importance. Our observations demonstrate that efficient penetration of epithelial barriers requires the cooperative activity of two Salmonella pathogenicity islands encoding different secretion systems. These findings underline the role of the epithelial brush border and reveal a new mechanism used by bacterial pathogens to overcome this barrier.