Abstract: During oxidative stress, cell apoptosis is promoted through the mitochondrial death pathway. Increased reactive oxygen species (ROS) are linked to excess cell loss and mediate the induction of apoptosis in various cell types. However, the role of ROS in the apoptotic pathway has not been clearly established. The aims of this study were to investigate the biochemical and morphological responses of rat astrocytes to hydrogen peroxide-mediated cell death and to define the role that melatonin might play in the apoptotic cascade. Hydrogen peroxide (H2O2; 0.1–1.0 mm) significantly reduced cell viability. Astrocyte death was associated with enhanced ROS production in a dose-dependent manner, as measured by 2′,7′-dichloro-fluorescein fluorescence. H2O2-induced cell death was found to be mediated through an apoptotic pathway as treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H2O2 also triggered caspase-3 activation and Bax expression. The ability of different antioxidants to prevent H2O2-induced apoptosis was examined by pre-incubating rat astrocytes with N-acetylcysteine (10 mm), glutathione (0.5 mm) or melatonin (0.1 mm and 10 nm). Results showed that N-acetylcysteine and glutathion can protect astrocytes against ROS accumulation and caspase-3 activation, whereas 0.1 mm melatonin can inhibit H2O2-induced apoptosis by regulating Bax expression and by inhibiting caspase-3 activation. Antiapoptotic effect of 10 nm melatonin associated to inhibition of Bax expression, give rise to new therapeutic approaches.