S100A4 protein, a member of the S100 superfamily of calcium-binding proteins, is frequently observed in various types of human cancers, including colorectal cancer (CRC). Our previous investigations have demonstrated that the overexpression of S100A4 is associated with lymph node metastasis and poor prognosis in CRC; however, its biological roles in CRC remain unclear. In the present study, we compared the expression of S100A4 at the mRNA and protein levels in six CRC cell lines, and found that the expression levels roughly coincided with their invasiveness. Using RNA interference, we suppressed S100A4 expression in SW620 CRC cells with highly invasive potential and S100A4 high expression. The specific knockdown of S100A4 strongly suppressed cell growth, migration and invasion activities. Furthermore, employing metastasis-related gene mRNA microarrays, we found four genes to be significantly dysregulated (more than 2-fold) after downregulation of S100A4, including three downregulated genes (MMP9, MMP10 and CDH11) and one upregulated gene (TIMP4). Our present results indicate that S100A4 may positively regulate tumor cell proliferation, invasion and metastasis associated with multiple molecules. Thus, the inhibition of S100A4 might be a potentially novel approach to treatment for CRC.