The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the Gαo/i-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. Gαo/i also activates the Src-Stat3 pathway. Here, we studied the relationship between the Gao/i-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from Gαo/i for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. Gαo-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through Gαo, mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that Gαo/i-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.