CD44 plays a major role in multiple physiological processes, including cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. It has been reported that highly expressed CD44 in certain types of tumors is associated with the hematogenic spread of tumor cells. The ability of CD44 to bind hyaluronan has been shown to correlate with tumor cell invasiveness, and it is likely that this ability may enhance tumor cell migration at several points during metastasis. However, the mechanism as to how CD44 stimulates metastasis remains unknown. The human breast tumor cell line, MDA-MB-435s, was used to investigate the effect of antibody-mediated CD44 crosslinking on the cellular level and localization of matrix metalloproteinase-9 (MMP-9). Confocal microscopy and immunocytochemical analyses were performed to demonstrate colocalization of CD44 and MMP-9 after CD44 crosslinking. Furthermore, the CD44-MMP-9 complex was purified by immunoprecipitation. G8 myoblast monolayers were employed to evaluate the invasiveness of human breast tumor cells after CD44 crosslinking in the presence or absence of protease inhibitors. CD44 crosslinking augmented the level of MMP-9 in the membrane of human breast tumor cells and clustering of CD44 serves as an MMP-9 docking molecule allowing MMP-9 to retain its concentrated proteolytic activity on the cell surface. Furthermore, crosslinking of CD44 enhances the ability of breast tumor cells to invade G8 myoblast monolayers and migrate through the basal membranes which was inhibited in the presence of anti-MMP-9 antibody or the MMP inhibitors GM6001 or 1,10-phenanthroline. This study demonstrates for the first time that CD44 crosslinking leads to an enhanced level and relocation of MMP-9 in human breast tumor cells accompanied by increased tumor invasion and metastasis.