Toll-like receptors (TLRs) are membrane-bound microbial sensors that mediate important host-to-microbe responses. Cell biology aspects of TLR function have been intensively studied in professional immune cells, in particular the macrophages and dendritic cells, but not well explored in other specialized epithelial cell types. The adult intestinal epithelial cells are in close contact with trillions of enteric microbes and engage in lifelong immune surveillance. Mature intestinal epithelial cells, in contrast to immune cells, are highly polarized. Recent studies suggest that distinct mechanisms may govern TLR traffic and compartmentalization in these specialized epithelial cells to establish and maintain precise signaling of individual TLRs. We, using immune cells as references, discuss here the shared and/or unique molecular machineries used by intestinal epithelial cells to control TLR transport, localization, processing, activation, and signaling. A better understanding of these mechanisms will certainly generate important insights into both the mechanism and potential intervention of leading digestive disorders, in particular inflammatory bowel diseases.