Abstract: Melatonin shows significant protective effects in Alzheimer’s disease (AD) models in vitro and in vivo; these effects are related to its function as an antioxidant. The source of reactive oxygen species (ROS) generation in the AD brain is primarily the amyloid-β (Aβ)- activated microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, the effects of melatonin on the activation of NADPH oxidase remain unclear. In the present study, the cultures of microglia were incubated in the presence of fibrillar Aβ1–42, which induces the assembly and the activation of NADPH oxidase, and triggers the production of superoxide anion-derived ROS. Pretreatment of microglia with melatonin dose-dependently prevents the activation of NADPH oxidase and decreases the production of ROS. Melatonin inhibits the phosphorylation of the p47phox subunit of NADPH oxidase via a PI3K/Akt-dependent signalling pathway, blocks the translocation of p47phox and p67phox subunit to the membrane, down-regulates the binding of p47phox to gp91phox, and impairs the assembly of NADPH oxidase. Our data offer new insights into the mechanism of inhibiting ROS generation by melatonin in Aβ-activated microglia. Inhibition of ROS production indirectly might be the underlying mechanism for the neuroprotection by melatonin in the AD brain.