Background: Brain-derived neurotrophic factor (BDNF) plays a crucial role in promoting survival and differentiation of neurons and neural stem cells (NSCs), but the downstream regulating mechanisms remain poorly understood. Objective: We investigated whether BDNF exerts its effect by triggering the phosphoinositide 3-kinase (PI3K), protein kinase B, PKB (AKT), glycogen synthase kinase-3 beta (GSK-3 beta) and beta-catenin signaling pathway in cultured neurons and NSCs derived from the rat embryonic spinal cord. Method: Immunocytochemistry was used to detect neuronal and NSCs characteristics. RT-PCR was used to detect PI3K/AKT/GSK3 beta/beta-catenin pathway expression. Results: Neurons and NSCs were successfully separated and cultured from Sprague-Dawley rat embryonic spinal cord and were respectively labeled using immunocytochemistry. Neuron-specific nuclear protein, neuronal class III beta-tubulin, and neurofilament expression were detected in neurons; nestin, glial fibrillary acidic protein, microtubule-associated protein 2 and chondroitin sulfate glycosaminoglycan expression were detected in the NSCs. BDNF promoted significant neuronal growth (number, soma size, and average neurite length), as well as NSCs proliferation and differentiation, but BDNF antibody decreased neuronal growth and NSCs proliferation and differentiation. RT-PCR was used to detect changes in BDNF signal pathway components, showing that BDNF upregulated tropomyosin receptor kinase B, phosphoinositide 3-kinase (PI3K), AKT and beta-catenin, but downregulated GSK-3 beta in the neurons and NSCs. BDNF antibody downregulated BDNF, tropomyosin receptor kinase B, PI3K, AKT, beta-catenin and cellular-myelocytomatosis viral oncogene, but upregulated GSK3, in the neurons and NSCs. Conclusion: Our findings suggested that BDNF contributed to neuronal growth and proliferation and differentiation of NSCs in vitro by stimulating PI3K/AKT/GSK3 beta/beta-catenin pathways.