Rho GTPase is a master regulator controlling cytoskeleton in multiple contexts such as cell migration, adhesion and cytokinesis. Of several Rho GTPases in mammals, the best characterized is the Rho subfamily including ubiquitously expressed RhoA and its homologs RhoB and RhoC. Upon binding GTP, Rho exerts its functions through downstream Rho effectors, such as ROCK, mDia, Citron, PKN, Rhophilin and Rhotekin. Until recently, our knowledge about functions of Rho and Rho effectors came mostly from in vitro studies utilizing cultured cells, and their physiological roles in vivo were largely unknown. However, gene-targeting studies of Rho and its effectors have now unraveled their tissue- and cell-specific roles and provide deeper insight into the physiological function of Rho signaling in vivo. In this article, we briefly describe previous studies of the function of Rho and its effectors in vitro and then review and discuss recent studies on knockout mice of Rho and its effectors.