NF-κB signaling pathway plays a critical role in cerebral ischemic stroke. MicroRNA-181b (miR-181b) induces the expression of NF-κB signaling pathways indirectly, and isosteviol sodium (STVNa) protects against ischemic injury via the inhibition of NF-κB-mediated inflammatory and apoptotic responses. However, the function of miR-181b and the actual relationship between STVNa and miR-181b in the ischemia-induced activation of NF-κB signaling pathways remains unclear. In this study, we found that miR-181b expression was significantly decreased in N2A neuroblastoma cells after CoCl-induced hypoxic injury in vitro. We further found, via western blot analysis and quantitative polymerase chain reaction assay, that altering miR-181b expression could induce changes in one of its target proteins, cylindromatosis (CYLD). Specifically, upregulation and downregulation of miR-181b (through transfection of either pre- or small interfering miR-181b, respectively) could negatively regulate CYLD protein levels as well as N2A cell survival rate and apoptosis following CoCl-induced injury. Furthermore, STVNa treatment following ischemic injury significantly downregulated the expression of miR-181b to alter apoptotic proteins downstream of the NF-κB signaling pathway through increasing CYLD protein levels in vivo and in vitro. STVNa also had a protective effect on CoCl-injured N2A cells, increasing cell survival rate, inhibiting apoptosis, reducing the damage of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Together, these results suggest that STVNa may downregulate miRNA-181b to protect mouse brain with ischemia stroke and against hypoxic injury in N2A cells by repressing NF-κB signaling pathways through the activation of CYLD, providing a novel therapy for ischemic stroke.