Recent studies suggest ex vivo modeling of neuronal injury is a robust approach for the mechanistic study of neurodegeneration. Melatonin, an indolamine, is a versatile molecule with antioxidative, antiapoptotic, neuroprotective, and anti-inflammatory properties. While melatonin has been studied as a therapeutic agent for spinal cord injury (SCI) related neuronal cell loss, its actions in organotypic slice cultures approximating SCI effects are less well understood. The actions of melatonin were therefore examined following exposure of cultured rat spinal cord slices to glutamate excitotoxicity. Exposure to glutamate (500 μM) for 4 hours induced neuronal degeneration that was prevented by 0.5 μM melatonin (applied immediately or 4 hours following glutamate exposure). Decreased internucleosomal DNA fragmentation, Bax:Bcl-2 and calpain:calpastatin ratios, caspase 8, 9 and 3 activities in slice cultures were measured following melatonin treatment. Melatonin receptor (MTR1, MTR2) mRNA levels were increased in the melatonin treated spinal cord slices. To confirm melatonin receptor-mediated protection, slice cultures were treated with 10 or 25 μM luzindole (melatonin receptor antagonist) at 0 and 4 hours, respectively, after glutamate exposure. Luzindole significantly decreased the ability of melatonin to prevent cell death in the sliced culture model. These results suggest melatonin receptors may provide a pathway for therapeutic applications to prevent penumbral neuron loss following SCI.