S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca(2+)-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines and further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. Our present results suggest the possibility that the inhibition of S100A4 can be utilized in antitumor applications for patients with pancreatic cancer.