Escherichia coli is a highly versatile and remarkably diverse organism. This bacterium is known as commensal that inhabits the gut of healthy people. However, they may acquire a combination of mobile genetic elements to become a highly adapted pathogen capable of causing intestinal or extraintestinal infections. In vitro and in vivo experiments have identified the existence of two novel pathotypes: translocating E. coli (TEC) that employ an unknown mechanism for translocating across the epithelium to the mesenteric lymph nodes and then to blood stream to cause septicaemia and adherent-invasive E. coli (AIEC) which are implicated as a microbiological aetiological factor in the pathogenesis of inflammatory bowel disease (IBD). Although the mechanism(s) by which TEC strains cause disease is unknown, recent genomic comparison of the TEC strain HMLN-1, and non-TEC strains representing all major E. coli pathotypes has shown the presence of the genomic island GI-argU, which encodes a Type 6 Secretion System (T6SS). We investigated the interaction of HMLN-1 strain with the gut epithelium using a co-culture of Caco-2 and HT29-MTX cells as a model of human intestinal epithelium. We also studied the expression of T6SS during its adhesion and invasion of the co-culture using clpV and vgrG gene targets. The HMLN-1 strain adhered to the co-culture cells in a diffusely adherent pattern and invaded and translocated significantly higher that all control strains. The gene expression studies revealed significant upregulation of T6SS genes clpV and vgrG during invasion and translocation compared with control strains suggesting a role for T6SS in pathogenesis of HMLN-1 and translocation across epithelial cells.