Melatonin has been widely studied as a protective agent against oxidative stress. However, the molecular mechanisms underlying neuroprotection in neurodegeneration and ischemic stroke are not yet well understood. In this study, we evaluated the neuroprotective/antioxidant mechanism of action of melatonin in organotypic hippocampal cultures (OHCs) as well as in photothrombotic stroke model in vivo. Melatonin (0.1, 1, and 10 μm) incubated postoxygen and glucose deprivation (OGD) showed a concentration-dependent protection; maximum protection was achieved at 10 μm (90% protection). Next, OHCs were exposed to 10 μm melatonin at different post-OGD times; the protective effect of melatonin was maintained at 0, 1, and 2 hr post-OGD treatment, but it was lost at 6 hr post-OGD. The protective effect of melatonin and the reduction in OGD-induced ROS were prevented by luzindole (melatonin antagonist) and α-bungarotoxin (α-Bgt, a selective α7 nAChR antagonist). In Nrf2 knockout mice, the protective effect of melatonin was reduced by 40% compared with controls. Melatonin, incubated 0, 1, and 2 hr post-OGD, increased the expression of heme oxygenase-1 (HO-1), and this overexpression was prevented by luzindole and α-bungarotoxin. Finally, administration of 15 mg/kg melatonin following the induction of photothrombotic stroke in vivo, reduced infarct size (50%), and improved motor skills; this effect was partially lost in 0.1 mg/kg methyllycaconitine (MLA, selective α7 nAChR antagonist)-treated mice. Taken together, these results demonstrate that postincubation of melatonin provides a protective effect that, at least in part, depends on nicotinic receptor activation and overexpression of HO-1.