Enterohaemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally-encoded 365 kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonisation of cattle by EHEC serogroup O5, O111 and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill-defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells, and to a lesser extent proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, 8, WC-1 and γδ-TCR) and cytokines examined (IL-2, -4, -10, -17A and IFN-γ), and rendered T cells refractory to mitogen for a least 18 hours after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavilin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB, L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.