The aim of this study was to test the hypothesis that EPEC infection causes molecular changes in host epithelial cells that predispose to neoplastic transformation. Model systems for EPEC infection were successfully established using in vitro co-culture with human colorectal cancer cell lines and co-culture with ex vivo human colonic mucosa; human adenocarcinomas were also probed for the presence of AE E. coli. Immunofluorescence identified mucosa associated AE E. coli in 5/20 (25%) adenocarcinomas. When co-cultured with normal human colonic mucosa, EPEC entered 10.6% of crypts, and closely associated with cells in the proliferative progenitor compartment. Mass spectrometry and microarray analysis validated our in vitro model and revealed a range of proteomic and transcriptomic effects in EPEC infected cells. Western blots and quantitative immunofluorescence demonstrated that EPEC downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 and the Wnt signalling/ adhesion protein β-catenin in vitro. Disruption of DNA mismatch repair is a causative factor in the development of many hereditary and sporadic colorectal cancers, and disruption of cell-cell adhesion has the potential to subvert normal colonic crypt homeostasis. These novel findings therefore suggest that chronic EPEC infection can predispose to cancer development by increasing the susceptibility of colonic epithelial cells to mutation by dietary or other carcinogens, and by altering expression of cytoskeletal and cell attachment proteins.