Neutrophil (PMN) transepithelial migration (TEM) and accumulation in luminal spaces is a hallmark of mucosal inflammation. TEM has been extensively modeled; however, the functional consequences and molecular basis of PMN interactions with luminal epithelial ligands are not clear. Here we report that cytokine-induced expression of a PMN ligand, intercellular adhesion molecule-1 (ICAM-1), exclusively on the luminal (apical) membrane of the intestinal epithelium results in accumulation and enhanced motility of transmigrated PMN on the apical epithelial surface. Using complementary in-vitro and in-vivo approaches, we demonstrate that ligation of epithelial ICAM-1 by PMN or with specific antibodies results in myosin light-chain kinase-dependent increases in epithelial permeability that are associated with enhanced PMN TEM. Effects of ICAM-1 ligation on epithelial permeability and PMN migration in vivo were blocked after intraluminal addition of peptides derived from the cytoplasmic domain of ICAM-1. These findings provide new evidence for functional interactions between PMN and epithelial cells after migration into the intestinal lumen. Although such interactions may aid in clearance of invading microorganisms by promoting PMN recruitment, engagement of ICAM-1 under pathologic conditions would increase accumulation of epithelial-associated PMN, thus contributing to mucosal injury as observed in conditions, including ulcerative colitis.