Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of animal and zoonotic pathogens of worldwide importance. Our previous research established that intestinal colonization of calves by EHEC serotypes O5 : H– and O111 : H– requires EHEC factor for adherence (Efa-1), also known as lymphostatin (LifA). Towards an understanding of the mode of action of Efa-1/LifA, chromosomal in-frame deletions of predicted glycosyltransferase (DXD) and cysteine protease (CHD) motifs were created in a Δstx1 derivative of EHEC O26 : H–. The magnitude and duration of faecal excretion of EHEC O26 : H– were significantly reduced by null mutation of efa-1/lifA, but were not impaired by ΔDXD or ΔCHD mutations, in contrast to observations made with truncated Efa-1/LifA mutants of Citrobacter rodentium in mice. Although C. rodentium Efa-1/LifA influences the induction of colonic hyperplasia in mice, EHEC O26 : H– Efa-1/LifA was not required for fluid accumulation or neutrophil recruitment in bovine ileal loops. In contrast to observations with EHEC O5 : H– or O111 : H– mutants, inactivation of efa-1/lifA in EHEC O26 : H– did not significantly affect adherence or secretion of type III secreted proteins that play pivotal roles in calf colonization. Lymphostatin activity could not be reliably demonstrated in lysates of EHEC O26 : H–; however, deletion of the glycosyltransferase and cysteine protease motifs in Efa-1/LifA from enteropathogenic E. coli O127 : H6 abolished lymphostatin activity. Our data uncouple the role of Efa-1/LifA in calf colonization from effects on type III secretion and reinforce the potential for pathotype- and serotype-specific phenotypes.