We compared a non-metastasising sarcoma cell population with three related populations of increasing metastatic potential. The metastatic cells in vitro exhibited a significantly reduced incidence of actin stress fibres but enhanced motility and chemotaxis. We also investigated gene expression underlying progression to a metastatic phenotype by performing a microarray analysis of the four sarcoma populations. We identified a subset of genes with significantly altered expression levels between non-metastasising and metastasising cells in tissue culture and in primary tumours. One such gene, encoding protein 4.1B, is downregulated in the metastatic sarcoma populations. To investigate possible roles of 4.1B in the mechanisms of metastasis, we used RNA interference (RNAi) to reduce its expression in the non-metastatic cells. Cells with reduced 4.1B expression displayed an altered F-actin morphology, with significantly fewer stress fibres. We also found that the 4.1B RNAi cells migrated at twice the speed of the untreated cells. Metastatic cells exogenously expressing 4.1B migrated at half the speed of control metastatic cells and displayed suppressed chemotaxis. Therefore, we propose that the reduction of 4.1B in the metastatic cells promotes the metastatic phenotype as a result of inducing a loss of actin stress fibres and a concomitant increase in cell motility.