Phosphatidylinositol 3-kinase (PI3K) plays several important roles in neuronal survival. Activation of the pathway is essential for the neuroprotective mechanisms of materials that shield neuronal cells from many stressful conditions. However, there have been no reports to date about the effect of the direct activation of the pathway in hypoxic injury of neuronal cells. We investigated whether the direct activation of the PI3K pathway inhibits neuronal cell death induced by hypoxia. Primary cultured cortical neurons (PCCNs) were exposed to hypoxic conditions (less than 1 mol% O2) and/or treated with PI3K activator. Hypoxia reduced the viability of PCCNs in a time-dependent manner, but treatment with PI3K significantly restored viability in a concentration-dependent manner. Among the signaling proteins involved in the PI3K pathway, those associated with survival, including Akt and glycogen synthase kinase-3β, were decreased shortly after exposure to hypoxia and those associated with cell death, including BAX, apoptosis-induced factor, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were increased. However, treatment with PI3K activator normalized the expression levels of those signaling proteins. PARP activity and levels of ATP and NAD(+) altered by hypoxia were also normalized with direct PI3K activation. All these findings suggest that direct and early activation is important for protecting neuronal cells from hypoxic injury.