Semaphorin molecules serve as axon guidance signals that regulate the navigation of neuronal growth cones. Semaphorins have also been implicated in other biological processes, including the immune response. Plexins, acting either alone or in complex with neuropilins, have recently been identified as functional semaphorin receptors. However, the mechanisms of signal transduction by plexins remain largely unknown. We have demonstrated a direct interaction between plexin-B1 and activated Rac. Rac specifically interacts with the cytosolic domain of plexin-B1, but not with that of plexin-A3 or -C1. Neither RhoA nor Cdc42 interacts with plexin-B1, indicating that the Rac/plexin-B1 interaction is highly specific. The binding of GTP and the integrity of the Rac effector domain are required for the interaction with plexin-B1. Furthermore, we have identified that a Cdc42/Rac interactive binding (CRIB) motif in the cytosolic domain of plexin-B1 is essential for its interaction with active Rac. We have also observed that the semaphorin CD100, a ligand for plexin-B1, stimulates the interaction between plexin-B1 and active Rac. Our results support a model by which activated Rac plays a role in mediating semaphorin signals, resulting in reorganization of actin cytoskeletal structure.