Semaphorins are implicated in glioma progression, although little is known about the underlying mechanisms. We have reported plexin-B3 expression in human gliomas, which upon stimulation by Sema5A causes significant inhibition of cell migration and invasion. The concomitant inactivation of Rac1 is of mechanistic importance because forced expression of constitutively active Rac1 abolishes these inhibitory effects. Furthermore, Sema5A induces prominent cell collapse and ramification of processes reminiscent of astrocytic morphology, which temporally associate with extensive disassembly of actin stress fibers and disruption of focal adhesions, followed by accumulation of actin patches in protrusions. Mechanistically, Sema5A induces transient protein kinase C (PKC) phosphorylation of fascin-1, which can reduce its actin-binding/bundling activities and temporally parallels its translocation from cell body to extending processes. PKC inhibition or fascin-1 knockdown is sufficient to abrogate Sema5A-induced morphological differentiation, whereas the process is hastened by forced expression of fascin-1. Intriguingly, Sema5A induces re-expression of glial fibrillary acidic protein (GFAP), which when silenced restricts differentiation of glioma cells to bipolar instead of multipolar morphology. Therefore, we hypothesize complementary functions of fascin-1 and GFAP in the early and late phases of Sema5A-induced astrocytic differentiation of gliomas, respectively. In summary, Sema5A and plexin-B3 impede motility but promote differentiation of human gliomas. These effects are plausibly compromised in high-grade human astrocytomas in which Sema5A expression is markedly reduced, hence leading to infiltrative and anaplastic characteristics. This is evident by increased invasiveness of glioma cells when endogenous Sema5A is silenced. Therefore, Sema5A and plexin-B3 represent potential novel targets in counteracting glioma progression.