Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-γ-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.