We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen–glucose deprivation (OGD). Diazoxide pre‐treatment induced delayed pre‐conditioning and almost entirely attenuated the OGD‐induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP‐sensitive potassium channel blocker 5‐hydroxydecanoate abolished the protective effect of diazoxide while the non‐selective K_ATP channel blocker glibenclamide did not. The non‐selective K_ATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese‐superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide‐pre‐conditioned neurons after OGD. In pre‐conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3‐nitropropionic acid also induced pre‐conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre‐conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase‐inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP‐sensitive potassium channels.