Neurite outgrowth is influenced by positive and negative signals that include the semaphorins, an important family of axonal outgrowth inhibitors. Here we report that the Rac GTPase activating protein (GAP)α2-chimaerin is involved in Semaphorin 3A (Sema 3A) signaling. In dorsal root ganglion neurons, Sema 3A-induced growth cone collapse was inhibited by α2-chimaerin mutated to eliminate GAP activity or interaction with phosphotyrosine. Activation of α2-chimaerin by phorbol ester caused growth cone collapse. Active α2-chimaerin interacts with collapsin response mediator protein-2 (CRMP-2) and cyclin-dependent kinase (Cdk) 5/p35 kinase through its SH2 and GAP domains, respectively. Cdk5 phosphorylates CRMP-2 at serine 522, possibly facilitating phosphorylation of serine 518 and threonine 514 by glycogen synthase kinase 3β (GSK3β), a kinase previously implicated in Sema 3A signaling. Phosphorylation of CRMP-2 serine 522 was essential for Sema 3A-induced growth cone collapse, which is dependent on Cdk5 but not Rho kinase activity. α2-chimaerin, like CRMP-2, can associate with the Sema 3A receptor. These results indicate that active α2-chimaerin Rac GAP, Cdk5/p35, and its substrate CRMP-2, are implicated in the dynamics of growth cone guidance initiated through Sema 3A signaling.