Squamous mucosal epithelial cells constitutively express calprotectin in the cytoplasm. To study how this antimicrobial protein complex confers epithelial resistance to invading bacteria, an epithelial cell line was stably transfected to express the calprotectin complex. Cells expressing calprotectin resist invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium. Calprotectin expression was accompanied by altered actin organization, increased α3 integrin expression, and spreading cell morphology. In this study, we assessed whether calprotectin expression affects bacterial binding and uptake. Threefold-fewer Listeria organisms bound to the surfaces of calprotectin-expressing cells, and 10-fold fewer were localized intracellularly by immunofluorescence. Similarly, fewer Salmonella organisms bound to cells expressing calprotectin. Calprotectin-expressing and sham-transfected cells showed similar levels of expression of surface E-cadherin and intracellular adhesion molecule 1 (ICAM-1) by flow cytometry. Calprotectin-expressing transfectants expressed calprotectin on the cell surface as well as in the cytosol. In conclusion, two bacterial pathogens showed reduced binding to calprotectin-expressing epithelial cells. Calprotectin-expressing cells appeared to have internalized disproportionately fewer Listeria organisms, suggesting that reduced binding and translocation supplemented direct antimicrobial effects in calprotectin-expressing cells.