Abstract: β-Amyloid (Aβ) is strongly involved in the pathogenesis of Alzheimer’s disease (AD), and mitochondria play an important role in neurodegenerative disorders. To determine whether any different effect of melatonin on cultured neurons treated with Aβ in vitro and which may be produced through its different action on mitochondria at different stages of culture, we investigated the damage of cultured rat hippocampal neurons mitochondrial function induced by Aβ in young neurons [days in vitro 10 (DIV 10)] and senescent neurons (DIV 25) and the protective effect of melatonin. Rat hippocampal neurons were incubated with amyloid-β peptide 25–35 (Aβ25-35) alone or pretreatment with melatonin. Cell viability, mitochondrial membrane potential (Δψm), ATP and the activity of the respiratory chain complexes were measured. Data showed that Aβ25-35 caused a reduction in Δψm, inhibited the activity of the respiratory chain complexes and led to ATP depletion, melatonin attenuated Aβ25-35-induced mitochondrial impairment in young neurons, whereas melatonin had no effect on Aβ25-35-induced mitochondrial damage in senescent neurons. These results demonstrate that melatonin has differential effect on Aβ25-35-induced mitochondrial dysfunction at different stages of culture and suggest that melatonin is useful for the prevention of AD, rather than treatment.