Enterotoxigenic Escherichia coli (ETEC) is a common cause of acute diarrhea in resource-poor settings. We report that some ETEC strains elicit a reduction in trans-epithelial electrical resistance (TER) in polarized T84 epithelial cell monolayers. The effect was irreversible up to 48 hours after a three-hour infection and was observed with heat-labile enterotoxin (LT)-producing strains, but not with heat-stable enterotoxin (ST)-producing strains. Using purified LT, a mutant with reduced ADP-ribosylating activity, and the LT-B subunit alone, we demonstrate that TER reduction requires a functional enterotoxin. Treatment of monolayers with LT or LT-producing strains of ETEC increases paracellular permeability to fluorescein isothiocyanate-dextran. Our data suggest that LT-producing ETEC strains may induce intestinal barrier dysfunction.