Microglial activation and inflammation are linked to neuronal damage in numerous neurodegenerative diseases. Microglia are activated in Parkinson’s disease, which is characterized by the selective and progressive loss of dopaminergic (DA) neurons. Recent evidence suggests that the microglial response to neuronal damage (i.e. reactive microgliosis) can actively induce neuronal death and fuel progressive neuronal loss. The mechanisms through which DA neuron injury activates microglia are poorly understood. Here, we address the novel possibility that μ calpain is a neuron-injury signal that activates microglia to further propagate DA neuron toxicity. Calpain is an intracellular protease that is upregulated in damaged DA neurons and is elevated in postmortem PD patient brains. Western blot analysis revealed that μ calpain was present in conditioned media from MPP⁺-treated cultures. μ Calpain was selectively neurotoxic to DA neurons when added to primary rat neuron-glia cultures. Addition of μ calpain to microglia-enriched cultures elicited both intracellular reactive oxygen species (ROS) and extracellular superoxide, which was abolished by the calpain inhibitor, calpeptin. Additionally, cultures from NADPH oxidase deficient mice, which are unable to produce extracellular superoxide, were insensitive to μ calpain-induced DA neurotoxicity, emphasizing the critical role of microglia-derived ROS. Further, inhibition of extracellular μ calpain with E64 attenuated MPP⁺-induced DA neurotoxicity. Here, we are the first to identify μ calpain as a soluble neuron injury factor released by damaged DA neurons, which is then toxic to additional DA neurons through microglia-generated oxidative insult.