Enterohaemorrhagic Escherichia coli (EHEC) are of great public health importance, causing gastroenteritis, haemorrhagic colitis and haemolytic uraemic syndrome. EHEC pathogenesis is linked to the formation of attaching/effacing (A/E) lesions on intestinal epithelial cells (IECs) characterised by intimate attachment, microvillous effacement and actin polymerisation. A/E lesions are formed following EHEC type III secretion (T3S) of the Translocated intimin receptor (Tir) into IECs, which enables intimate attachment via binding to the adhesin intimin. Whereas EHEC colonisation in certain animal models has been demonstrated in the small intestine and the colon, clinical evidence of EHEC adherence to the human intestine is lacking. This is surprising as EHEC causes pathology and inflammation in the colon. In this study, we have used in vitro and ex vivo models to investigate EHEC colonisation of human IECs and the innate immune response to infection. In vitro organ culture (IVOC) of human endoscopic biopsies with EHEC revealed extensive colonisation of terminal ileum and the colon. Colonic colonisation was associated with microvillous effacement and Tir translocation, and was abrogated by deletion of genes encoding intimin as well as T3SS proteins EspA and EscN. In contrast, EHEC colonisation of T84 colon carcinoma cells was associated with microvillous effacement but not Tir translocation or actin recruitment, and occurred independently of intimin and the T3SS. EHEC infection of T84 cells resulted in an increase in expression of neutrophil chemokine interleukin-8 (IL-8) and antimicrobial peptide human β-defensin (hBD)-2, which was dependent on bacterial expression of the gene encoding flagellin. IVOC of colonic biopsies with EHEC also resulted in up-regulation of IL-8 and hBD2. These results indicate that EHEC colonises human colonic epithelium, triggering A/E lesion formation and activation of innate immune genes. These interactions likely contribute to colonic pathogenesis during infection and may uncover novel targets for anti-EHEC therapies.