Docosahexaenoic acid (DHA) has been shown to promote neuronal differentiation of neural stem cells (NSCs) in vivo and in vitro. Previously, we found that N-docosahexaenoylethanolamine (synaptamide), an endogenous DHA metabolite with an endocannabinoid-like structure, promotes neurite growth, synaptogenesis, and synaptic function. In this study, we demonstrate that synaptamide potently induces neuronal differentiation of NSCs. Differentiating NSCs were capable of synthesizing synaptamide from DHA. Treatment of NSCs with synaptamide at low nanomolar concentrations significantly increased the number of MAP2 and Tuj-1-positive neurons with concomitant induction of protein kinase A (PKA)/cAMP response element binding protein (CREB) phosphorylation. Conversely, PKA inhibitors or PKA knockdown abolished the synaptamide-induced neuronal differentiation of NSCs. URB597, a fatty acid amide hydrolase (FAAH) inhibitor, elevated the level of DHA-derived synaptamide and further potentiated the DHA- or synaptamide-induced neuronal differentiation of NSCs. Similarly, NSCs obtained from FAAH KO mice exhibited greater capacity to induce neuronal differentiation in response to DHA or synaptamide compared to the wild type NSCs. Neither synaptamide nor DHA affected NSC differentiation into GFAP-positive glia cells. These results suggest that endogenously produced synaptamide is a potent mediator for neurogenic differentiation of NSCs acting through PKA/CREB activation.