: As a potent free radical scavenger and antioxidant, melatonin protects brain tissue against ischemia–reperfusion injury, partly via suppression of ischemia‐induced production of nitric oxide, when given before ischemia–reperfusion or within 2 hr of onset of ischemia. In this study, we examined the neuroprotective effect of melatonin in an in vitro model of ischemia. Primary cultured astrocytes were subjected to 4 or 8 hr of oxygen–glucose deprivation (OGD), and cultured SHSY5Y human neuronal cells were exposed to 1 hr of OGD. Melatonin was added to the medium at the commencement of OGD to achieve different final concentrations, and cell death was quantified using the measurement of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) at 24 hr after reversion of OGD. Treatment with melatonin did not affect the astrocytic cell death following 4 or 8 hr of OGD. The relative MTT values of the neuronal cells were (as mean ± S.E.M.) 59.1 ± 2.4% in the vehicle‐treated OGD group and 80.1 ± 2.7%, 82.5 ± 2.9%, 74.1 ± 2.3%, 64.2 ± 2.3%, 62.7 ± 2.8%, and 61.0 ± 3.9% in the OGD groups treated with melatonin at 10−3, 10−4, 10−5, 10−6, 10−7, and 10−8 m, respectively. Reduction in cell death was significant following treatment with melatonin at 10−3, 10−4, or 10−5 m. Reverse transcription‐polymerase chain reaction showed that human mt1 and MT2 membrane receptors were not expressed in the cultured neuronal cells. Our results show that melatonin co‐treatment protects cultured neuronal cells but not astrocytes against OGD‐induced cell death in a dose‐dependent manner and that the neuroprotection is independent of its known membrane receptors.