We examined the effects of diazoxide, the putative mitochondrial adenosine triphosphate‐sensitive potassium (mitoK_ATP) channel opener, against glutamate excitotoxicity in primary cultures of rat cortical neurons. Cells were treated with diazoxide for 24 hr and then exposed to 200 μM glutamate. Cell viability was measured 24 hr after glutamate exposure. We found that treatment 24 hr before glutamate exposure with 250 and 500 μM diazoxide but not with another mitoK_ATP channel opener, nicorandil, increased neuronal viability from 54 ± 2% to 84 ± 2% and 92 ± 3%, respectively (n = 25–40). These effects were not inhibited by the putative mitoK_ATP channel blocker 5‐hydroxydecanoic acid. Diazoxide application increased production of reactive oxygen species (ROS) and coapplication of M40401, a superoxide dismutase mimetic, prevented delayed preconditioning. The 24 hr preconditioned neurons showed significantly reduced ROS production upon glutamate stimulation compared to that in untreated cells. These results suggest that diazoxide induces delayed preconditioning in cultured cortical neurons via increased ROS production and attenuation of oxidative stress upon glutamate stimulation. © 2004 Wiley‐Liss, Inc.