Metastasis, the major cause of deaths of cancer patients, is a complex multi-step process in which cell motility plays an important role. The motility related protein-1, MRP-1/CD9 (or CD9), is a tetraspanin membrane glycoprotein and it has been implicated in playing roles in cell adhesion, spreading, and motility. In this study, a replication-deficient recombinant adenovirus expressing CD9 (Ad-CD9) was generated. By using a highly metastatic breast cancer cell line JygMC(A) as the model, we evaluaed the effects of adenoviral-mediated CD9 expression on suppression of tumor metastasis and related biological and biochemical parameters. By comparing with untreated or control virus-treated cells, Ad-CD9 transduced JygMC(A) cells had an increased cell motility and increased adhesion to laminin and fibronectin but unchanged adhesion to matrigel. Overexpression of CD9 in JygMC(A) cells suppressed cell growth and decreased in vitro invasion ability of cells to penetrate matrigel. Moreover, Ad-CD9 transduced JygMC(A) cells formed less metastasis in the lungs and livers in nude mice than control groups in a spontaneous metastasis animal model. These results suggest that CD9 may act as a tumor suppressor and/or anti-metastasis gene. Adenoviral-mediated CD9 gene transfer may have a potential for cancer gene therapy to suppress tumor growth and metastases.