Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen‐infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild‐type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption.