In previous studies, we have shown that numbers of S100 calcium-binding proteins (including S100A4) are expressed differentially in astrocytic tumors according to their levels of malignancy. S100A4 is involved in tumor progression, cell migration and metastasis. This protein is able to play extracellular roles such as neuritogenic and angiogenic activities. The present study aims to investigate the possible role played by extracellular S100A4 in the in vitro migration of astrocytic tumor cells. The speed and rate of migration of living cells were measured using computer-assisted videomicroscopy. In parallel, we also analyzed the effects of extracellular S100A4 on the organization of the actin cytoskeleton and the expression of a number of its molecular regulators. These included small Rho-GTPases (RhoA, Rac1 and Cdc42) and some of their direct effectors (mDia and N-WASP), and also actin-binding proteins such as profilin and alpha-actinin. Our data demonstrate the influence of S100A4 on astrocytic tumor cells with respect to these different aspects. Indeed, we show that extracellular S100A4 treatments decrease both the amount of polymerized F-actin and the levels of expression of RhoA, mDia and profilin. While a decrease in the Cdc42 and N-WASP expression was also observed, the Rac1 expression remained unchanged. All these activities, which result in the stimulation of cell motility, contribute to the understanding of the extracellular role of S100A4.