Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli are non‐invasive attaching/effacing (A/E) bacterial pathogens that infect their host’s intestinal epithelium, causing severe diarrhoeal disease. These bacteria utilize a type III secretion apparatus to deliver effector molecules into host cells, subverting cellular function. Mitochondrial associated protein (Map) is a multifunctional effector protein that targets host cell mitochondria and contributes to infection‐induced epithelial barrier dysfunction in vitro. Unfortunately, the relevance of these actions to the pathogenesis of EPEC‐induced disease is uncertain. Using Citrobacter rodentium, a mouse‐adapted A/E bacterium, we found that Map colocalized with host cell mitochondria, and that in vivo infection led to a disruption of mitochondrial morphology in infected colonocytes as assessed by electron microscopy. Histochemical staining for the mitochondrial enzyme succinate dehydrogenase also revealed a significant loss of mitochondrial respiratory function in the infected intestinal epithelium; however, both pathologies were attenuated in mice infected with a Δmap strain. C. rodentium Map was also implicated in the disruption of epithelial barrier function both in vitro and in vivo. These studies thus advance our understanding of how A/E pathogens subvert host cell functions and cause disease, demonstrating that Map contributes to the functional disruption of the intestinal epithelium during enteric infection by C. rodentium.