Co-existing paracellular and transcellular barrier defect in intestinal epithelium was documented in inflammatory bowel disease, celiac disease, and intestinal obstruction. Mechanisms regarding tight junction disruption have been extensively studied; however, limited progress has been made in research on bacterial transcytosis. Densely packed brush border (BB), with cholesterol-based lipid rafts in the intermicrovillous membrane invagination, serves as an ultrastructural barrier to prevent direct contact of luminal microbes with the cellular soma. Evidence in in vitro epithelial cell cultures and in vivo animal models of bowel obstruction and antibiotic-resistant bacterial infection had indicated that nonpathogenic, noninvasive enteric bacteria may hijack the lipid raft-mediated endocytic pathways. Our studies have shown that low dose interferon-gamma (IFNγ) causes long myosin light chain kinase (MLCK)-dependent terminal web (TW) contraction and BB fanning, allowing bacteria to pass through the consequently widened intermicrovillous cleft to be endocytosed via caveolin-associated lipid rafts. Activation of intracellular innate immune receptors by bacteria-containing endosomes may further induce inflammatory and oxidative stress, leading to secondary tight junction damage. The finding of bacterial internalization preceding tight junction damage suggests that abnormal bacterial uptake by epithelial cells may contribute to the initiation or relapse of chronic intestinal inflammation.