Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid-β (Aβ) peptide in brain regions that are important for memory and cognition. The buildup of Aβ aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Aβ-induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 μm of Aβ25–35 in the absence or in the presence of melatonin (25, 50, or 100 μm). In addition, the authors have investigated the involvement of GSK-3β, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Aβ-induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Aβ25–35. In addition, melatonin significantly reduced the activation of GSK-3β, the phosphorylation of tau protein, the glial activation and the Aβ-induced increase of TNF-α and IL-6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Aβ-induced phosphorylation of tau protein, and preventing GSK-3β activation and neuroinflammation.