Colonization of the vaginal introitus by fecal Escherichia coli is thought to be a key initial event leading to acute urinary tract infection, yet the mannosylated receptor for type 1 pili on the squamous epithelium of vaginal mucosa is unknown. E. coli expressing type 1 pili adhered to sections of normal human vaginal epithelium in a gradient with greatest binding in upper cell layers was observed, which suggests that epithelial differentiation influences bacterial binding. Consistent with this observation, bacterial binding was enhanced in vaginal epithelial cultures that were induced to differentiate, and this enhanced bacterial binding was associated with increased K13 expression levels and increased binding of the mannose-specific lectin Galanthus nivalis agglutinin. These results demonstrate that the binding of type 1-piliated E. coli to vaginal epithelial cells correlates with epithelial differentiation and suggest that the vaginal receptor for type 1 pili is up-regulated during differentiation.