CD9, a transmembrane protein known as motility-related protein-1, plays a pivotal role in regulating cell adhesion, motility, and proliferation, and has been regarded as an important metastasis-inhibitory factor of various human cancers. However, little information has been obtained regarding the highly metastatic human small-cell lung cancer (SCLC). In the present study, an SCLC cell line (OS3-R5), lacking CD9 expression, was transfected with human CD9 gene to assess the role of CD9 on the metastatic potential of SCLC. CD9 gene transfection into OS3-R5 cells resulted in cell proliferation and motility in vitro. Parental and mock-transfected OS3-R5 cells developed liver metastasis and malignant ascites when they were intravenously inoculated into NK cell-depleted SCID mice. CD9 gene transfection into OS3-R5 cells caused suppression of the liver metastasis and malignant ascites. Immunohistochemical analysis revealed that the number of proliferating tumor cells was significantly fewer in liver lesions produced by CD9 gene-transfected OS3-R5 cells than those produced by parental or mock control OS3-R5 cells. In addition, no detectable levels of CD9 were expressed in metastatic tumor cells in mice bearing CD9 gene-transfected OS3-R5 cells, as well as those in mice bearing parental or mock control OS3-R5 cells. These results suggest that the restored expression of CD9 in SCLC cells may reduce the metastatic spread of SCLC cells via the inhibition of cell proliferation and motility.