The Gαo/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through Gαo/i to induce neurite outgrowth were studied. The expression of Gαo/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. Gαo/i- induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. Gαo/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Hap1GAPII also blocks the Gαo/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous Gαo/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the Gαo/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth. © 2005 by The American Society for Biochemistty and Molecular Biology, Inc.