The Rho-LIM-kinase (LIMK) signaling pathway, believed to be involved in the regulation of tumor invasion, specifically regulates the activity of cofilin. However, it is unclear whether cofilin plays a pivotal role in tumor invasiveness. In this paper we show using a tet-on gene expression system that over-expression of cofilin inhibits the invasiveness of human lung cancer H1299 cells. Over-expressed cofilin disrupts the actin cytoskeleton at the leading edge of the cell and up-regulates p27(kip1), which is known to be involved in regulating cell motility. Removal of cofilin over-expression normalizes the p27(kip1) level and concomitantly restores the invasiveness of the cultured cells. These findings suggest that excessive cofilin production might prevent cancer cell invasion.