The human adenoid epithelium forms a continuous barrier against a wide variety of exogenous antigens. In this study, to elucidate the structures of the epithelial barrier in the human adenoid, including M-cells, we identified M-cells using an anti-cytokeratin 20 (Ck20) antibody and investigated expression of tight junction proteins in human adenoid epithelium in vivo and in vitro. In human adenoid epithelium and primary cultures, mRNAs of occludin, junctional adhesion molecule-A, ZO-1, and claudin-1, -4, -7, and -8 were detected by reverse transcription-polymerase chain reaction, whereas claudin-2 and -9 were expressed in vitro. In the epithelium in vivo, some Ck20-positive cells were randomly observed and indicated pocket-like structures, whereas Ck7 was positive in almost cells. Transmission electron microscopy revealed that Ck20-associated gold particles could be identified in M-like cells which had short microvilli and harboured the lymphocyte in the pocket-like structure. In primary cultures in vitro, Ck20-positive cells were also detected and had a function to take up fluorescent microparticles. In Ck20-positive cells in vivo and in vitro, expression of occludin, ZO-1, claudin-1 and -7 were observed at cell borders. These results indicate that the epithelial barrier of the human adenoid is stably maintained by expression of tight junction proteins in the epithelium including Ck20-positive M-like cells.