Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild-type EPEC Tir and the non-tyrosine-phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.