Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces microglial activation and loss of dopaminergic neurons in models of Parkinson's disease. However, the mechanisms of rotenone neurotoxicity are still poorly defined. Here, we used primary neuronal/glial cultures prepared from rat cerebella to investigate the contribution of microglia to neuronal cell death induced by low concentrations of rotenone. Rotenone at 2.5 nm induced neuronal loss over several days without increasing the numbers of necrotic or apoptotic neurons, and neuronal loss/death could be prevented by selective removal of microglia. Rotenone increased microglial proliferation and phagocytic activity, without increasing tumour necrosis factor-α release. Rotenone-induced neuronal loss/death could be prevented by inhibition of phagocytic signalling between neurons and microglia with: cyclo(Arg-Gly-Asp-d-Phe-Val) (to block the microglial vitronectin receptor); MRS2578 (to block the microglial P2Y6 receptor); or either annexin V or an antibody against phosphatidylserine (to block exposed phosphatidylserine, a well-characterized neuronal ‘eat-me’ signal). As inhibition of phagocytosis by five different means prevented neuronal loss without increasing neuronal death, these data indicate that rotenone neurotoxicity is at least partially mediated by microglial phagocytosis of otherwise viable neurons (phagoptosis). Thus, neuronal loss in Parkinson's disease and other neurological diseases might be prevented by blocking phagocytic signalling.