Stevioside, isolated from the herb Stevia rebaudiana, has been widely used as a food sweetener all over the world. Isosteviol Sodium (STV-Na), an injectable formulation of isosteviol sodium salt, has been proved to possess much greater solubility and bioavailability and exhibit protective effects against cerebral ischemia injury in vivo by inhibiting neuron apoptosis. However, the underlying mechanisms of the neuroprotective effects STV-Na are still not completely known. In the present study, we investigated the effects of STV-Na on neuronal cell death caused by hypoxia in vitro and its underlying mechanisms.We used cobalt chloride (CoCl) to expose mouse neuroblastoma N2a cells to hypoxic conditions in vitro.Our results showed that pretreatment with STV-Na (20 μM) significantly attenuated the decrease of cell viability, lactate dehydrogenase release and cell apoptosis under conditions of CoCl-induced hypoxia. Meanwhile, STV-Na pretreatment significantly attenuated the upregulation of intracellular Ca concentration and reactive oxygen species production, and inhibited mitochondrial depolarization in N2a cells under conditions of CoCl-induced hypoxia. Furthermore, STV-Na pretreatment significantly downregulated expressions of nitric oxide synthase, interleukin-1β, tumor necrosis factor-α, interleukin-6, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) signalings in N2a cells under conditions of CoCl-induced hypoxia.Taken together, STV-Na protects neural cells against hypoxia-induced apoptosis through inhibiting MAPK and NF-κB pathways.