Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.