Melatonin (MEL) and its metabolites serve as endogenous reactive oxygen species (ROS) scavengers and have a wide spectrum of antioxidant activity. Cobalt chloride is one of the commonly used hypoxia-mimetic agents, due to blocking the degradation of and triggering the accumulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein, which is very well known as a critical regulator of the cellular response against hypoxia. In the current study we aimed to determine the possible protective effects of melatonin on a cobalt chloride-induced hypoxia model of SH-SY5Y neuronal cells. Group I was the control group and SH-SY5Y cells were incubated in normal culture media without any chemical administration. In Group II, SH-SY5Y cells were incubated with 1 mu M MEL for 24 h. In Group III cells were incubated with 200 mu M cobalt chloride for 24 h. The last group, Group 4, was a combination group of cobalt chloride and MEL. Cells were preincubated with 1 mu M for 12 h and then 200 mu M cobalt chloride for 24 h. We performed the cell viability test (MTT) and checked the caspase-3 and -9 activities and the lipid peroxidation (LP), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), mitochondrial depolarization, and intracellular ROS levels. We observed that cobalt chloride increased intracellular ROS, caspase-3 and -9, lipid peroxidation, and mitochondrial membrane depolarization values, while decreasing GSH levels and GSH-Px activity. However, GSH and GSH-Px values were increased by melatonin treatment although lipid peroxidation level, intracellular ROS production, and caspase-3 and -9 activities were decreased by the treatment. In conclusion, we observed that melatonin incubation protects neuronal cells against hypoxia-induced oxidative stress.