Semaphorins and plexins are implicated in the progression of various types of cancer, although the molecular basis has not been fully elucidated. Here, we report the expression of plexin-B3 in glioma cells, which upon stimulation by its ligand Sema5A results in significant inhibition of cell migration and invasion. A search for the underlying mechanism revealed direct interaction of plexin-B3 with RhoGDP dissociation inhibitor α (RhoGDIα), a negative regulator of RhoGTPases that blocks guanine nucleotide exchange and sequesters them away from the plasma membrane. Glioma cells challenged with Sema5A indeed showed a marked reduction in Rac1-GTP levels by 60%, with a concomitant disruption of lamellipodia. The inactivation of Rac1 was corroborated to contribute to the impediment of glioma cell invasion by Sema5A, as supported by the abolishment of effect upon forced expression of a constitutively active Rac1 mutant. Furthermore, silencing the endogenous expression of RhoGDIα in glioma cells was found to be sufficient in abrogating the down-regulation of Rac1-GTP and the ensuing suppression of glioma cell motility induced by Sema5A. Mechanistically, we provide evidence that Sema5A promotes Rac1 recruitment to RhoGDIα and reduces its membrane localization in a plexin-B3-dependent manner, thereby preventing Rac1 activation. This represents a novel signaling of semaphorin and plexin in the control of cell motility by indirect inactivation of Rac1 through RhoGDIα.