Translocation ofPseudomonas aeruginosathrough epithelial tissues can cause sepsis. Here, we examined whetherP. aeruginosapenetrates epithelial cell layers using type IV pili (TFP). Deletion of TFP (pilA) did not affect association with Caco-2 cells, although it decreased penetration through, and disruption of, Caco-2 cell monolayers. We found that TFP are necessary for injection of the type III effector ExoS, which impairs defense againstP. aeruginosapenetration, into host cells. Deletion ofpilAattenuated oral infection in silkworms. We conclude thatP. aeruginosainjects ExoS into cells through the function of TFP, enabling penetration of epithelial barriers.