Aim: To develop a new adherence assay, using cattle recto-anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Methods and Results: Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp-2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae-independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. Conclusion: The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp-2 cell adherence assay. Significance and Impact of the study: The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto-anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food-borne pathogens that colonize cattle via adherence to this particular anatomical site.