Patients with inflammatory bone disease or cancer have an increased risk of fractures and delayed healing of bones. S100A4 protein is a member of the calcium-binding S100 protein family, which is abundantly expressed in inflammatory diseases and cancers. In this study, we investigated the effects of extracellular S100A4 on osteoblasts, cells responsible for bone formation. Treatment of mouse primary calvarial osteoblasts with recombinant S100A4 resulted in reductions in matrix mineralization. The expression of osteoblast marker genes including osteocalcin and osterix was also suppressed. Interestingly, S100A4 stimulated the nuclear factor-kappaB (NF-κB) signaling pathway in osteoblasts. More importantly, ex vivo organ culture of mouse calvariae with recombinant S100A4 decreased the expression levels of osteocalcin, supporting the results of our in vitro experiments. Our findings suggest that extracellular S100A4 plays an important role in the regulation of bone formation by activating the NF-κB signaling pathway in osteoblasts.