Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.