Efficient downregulation of CXCR4 cell surface expression by introduction of the CD63 gene has previously been reported by us. In the present study, it was found that CD63 and its mutant efficiently interact with CXCR4 in live cells and that CD63-induced downregulation and interaction are significantly abrogated by the N-linked glycosylation inhibitor, TM. Furthermore, the downregulation and interaction were clearly attenuated by alternation of all three N-linked glycosylation sites in CD63. Either CD63 or CD63 Delta N formed a complex with CXCR4 at the Golgi apparatus and the late endosomes, while CD63 GD mutants lost the ability to forma complex with CXCR4 exclusively at the Golgi apparatus. These findings suggest that CD63 interacts with CXCR4 through the N-linked glycans-portion of the CD63 protein and that the complex induces direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. At the Golgi apparatus, there may be lysosome protein (CD63)-associated machinery that influences trafficking of other membrane proteins.