Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of mitochondrial fission protein dynamin-related protein 1 (Drp1), has been reported to display neuroprotective properties in different animal models. In the present study, we investigated the protective effect of mdivi-1 on β-amyloid protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that mitochondrial fission was increased in Aβ treatment and inhibition of mitochondrial fission by mdivi-1 significantly reduced Aβ-induced expression of CD11b (a marker of microglial activation), viability loss and apoptotic rate increase in BV-2 and primary microglial cells. Moreover, we also found that mdivi-1 treatment markedly reversed mitochondrial membrane potential loss, cytochrome c (CytC) release and caspase-3 activation. Altogether, our data suggested that mdivi-1 exerts neuroprotective effects against Aβ-induced microglial apoptosis, and the underlying mechanism may be through inhibiting mitochondrial membrane potential loss, CytC release and suppression of the mitochondrial apoptosis pathway.