Bacterial flagellin activates innate immune responses by signaling through Toll-like receptor 5 and is a potential vaccine adjuvant. Mucosal lymphoid follicles, inductive sites for adaptive mucosal immune responses, are covered by a follicle-associated epithelium (FAE) specialized for the uptake of antigens. This study demonstrates that mucosal application of Salmonella dublin flagellin enhanced transepithelial transport of microparticles by the FAE of mouse Peyer's patches in vivo. Flagellin also induced rapid, matrix metalloproteinase-dependent migration of subepithelial dendritic cells (DCs) into the FAE, better positioning DCs for antigen capture. These innate responses to flagellin enhance FAE functions and may promote adaptive immune responses in the mucosa.