Technical Abstract: Background and aims: Mucosa-associated E. coli are abundant in Crohn’s disease (CD) but whether these bacteria gain intracellular access within the mucosa is less certain. If E. coli does gain intracellular access in CD, the contribution of bacterial pathogenicity as opposed to a defect in host innate immunity to this requires further elucidation. This study aimed to quantify and characterise mucosa-associated and intracellular E. coli in CD and controls. Methods: Mucosal biopsies from 30 patients with CD, 15 with ulcerative colitis (UC) and 14 healthy controls were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli respectively. Forty of these strains (CD (n = 24), UC (n = 9) and HC (n = 7)) were characterised by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance(AMR)genes and proteomic analysis. Results: Mucosa-associated E. coli were more abundant in CD and UC than healthy controls (2750 vs. 1350 vs. 230 median cfu/biopsy, P = .01). Intracellular E. coli were more prevalent in CD (90%) than UC (47%) or healthy control mucosal biopsies (0%)(P < .001). Extraintestinal pathogenic E. coli virulence factors and AMR genes were identified in E. coli strains from all 3 groups. 2/24 CD strains were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC and healthy control strains, or intracellular isolates from mucosa-associated isolates. Conclusion: Intracellular E. coli are more common in CD than UC or healthy controls. Most intracellular CD E. coli did not have characterising pathogenic features, thus suggesting a greater role for defective mucosal immunity in their ability to gain intracellular access.