Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.