Rho family proteins regulate multiple cellular functions including motility and invasion through regulation of the actin cytoskeleton and gene expression. Activation of Rho proteins is controlled precisely by multiple regulators in a spatiotemporal manner. RhoA and/or RhoC are key players that regulate the metastatic activity of malignant tumor cells, and it is therefore of particular interest to understand how activation of these Rho proteins is controlled. We recently identified an upstream regulator of RhoA activation, p27RF-Rho (p27(kip1) releasing factor from RhoA) that acts by freeing RhoA from inhibition by p27(kip1). p27(kip1) is a cell cycle regulator when it is localized to the nucleus, but it binds RhoA and inhibits activation of the latter when it is localized to the cytoplasm. Here, we show that a metastatic variant of mouse melanoma B16 cells (F10) exhibits greater expression of p27RF-Rho, RhoA, and RhoC than the nonmetastatic parental cells (F0). Injection of F10 cells into mouse tail vein resulted in the formation of metastatic lung colonies, whereas prior knockdown of expression of either one of the three proteins using specific shRNA sequences decreased metastasis markedly. p27RF-Rho regulated the activation of RhoA and RhoC and thereby modulated cellular adhesion and motility, in addition to pericellular proteolysis. The Rho activities enhanced by p27RF-Rho had a marked effect upon efficiency of lodging of F10 cells in the lung, which represents an early step of metastasis. p27RF-Rho also regulated metastasis of human melanoma and fibrosarcoma cells. Thus, p27RF-Rho is a key upstream regulator of RhoA and RhoC that controls spreading of tumor cells.