Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are important human pathogens that rely on translocation of type III secretion system (T3SS) effectors for subversion of signal transduction pathways and colonization of the mammalian gut mucosa. While a core set of effectors is conserved between EPEC and EHEC strains, a growing number of accessory effectors that were found at various frequencies in clinical and environmental isolates have been recently identified. Recent genome projects identified espV as a pseudogene in EHEC but a putative functional gene in EPEC strains E110019 and E22 and the closely related mouse pathogen Citrobacter rodentium. The aim of this study was to determine the distribution of espV among clinical EPEC and EHEC strains and to investigate its function and role in pathogenesis. espV was found in 16% of the tested strains. While deletion of espV from C. rodentium did not affect colonization dynamics or fitness in mixed infections, expression of EspV in mammalian cells led to drastic morphological alterations, which were characterized by nuclear condensation, cell rounding, and formation of dendrite-like projections. Expression of EspV in yeast resulted in a dramatic increase in cell size and irreversible growth arrest. Although the role of EspV in infection and its target host cell protein(s) require further investigation, the data point to a novel mechanism by which the T3SS subverts cell signaling.