S100A4, a 10-12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.