Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is a key mediator of inflammation and stress in the central nervous system (CNS), and is highly expressed in the developing brain. In this study, we investigated the possible role of IL-1β in neuronal differentiation of cortical neural precursor cells (NPCs). We showed that stimulation with IL-1β increased expression levels of neurotrophin-3 (NT3) and neurogenin 1 (Ngn1) and promoted neurite outgrowth. We also found that IL-1β increased mRNA and protein levels of Wnt5a. Knockdown of Wnt5a by transfection with Wnt5a siRNA inhibited IL-1β-induced neuronal differentiation. Moreover, IL-1β-induced Wnt5a expression was regulated by nuclear factor kappa B (NF-κB) activation, which is involved in IL-1β-mediated neuronal differentiation. To examine the role of Wnt5a in neuronal differentiation of NPCs, we exogenously added Wnt5a. We found that exogenous Wnt5a promotes neuronal differentiation, and activates the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway. In addition, Wnt5a-induced neuronal differentiation was blocked by RhoA siRNA, as well as by a specific Rho-kinase inhibitor (Y27632) or a SAPK/JNK inhibitor (SP600125). Furthermore, treatment with RhoA siRNA, Y27632, or SP600125 suppressed the IL-1β-induced neuronal differentiation. Therefore, these results suggest that the sequential Wnt5a/RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation of NPCs.