The tripartite complex AcrAB-TolC is the major RND pump inEscherichia coliand other Enterobacteriaceae, includingShigella, the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes toShigella flexneriinvasion of epithelial cells. We found that deletion of bothacrAandacrBgenes causes reduced survival ofS. flexneriM90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such asShigella, and add insights into the mechanism governing theShigellainfection process.