Growing evidence suggest that microglia may play an important role in the pathogenesis of neurodegenerative disease including Parkinson's disease, Alzheimer's disease, and so forth. The activation of microglia may cause neuronal damage through the release of reactive oxygen species and proinflammatory cytokines. However, the early response of microglial cells remains unclear before cells can secrete the proinflammatory cytokines. Here, a time course analysis showed the earliest expression of inducible nitric oxide synthase and cyclooxygenase-2 at 3 and 24 h following lipopolysaccharide (LPS) treatment. To further define initial response proteins of microglia after LPS treatment, we utilized a novel mass spectrometry-based quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of the cell culture-conditioned media of 1 h LPS-treated microglia as compared with controls. The proteomic analysis identified 77 secreted proteins using SignalP; of these, 28 proteins were associated with lysosome of cells and 13 lysosome-related proteins displayed significant changes in the relative abundance after 1 h LPS treatment. Four proteins were further evaluated with Western blot, demonstrating good agreement with quantitative proteomic data. These results suggested that microglia first released some lysosomal enzymes which may be involved in neuronal damage process. Furthermore, ammonium chloride, which inhibits microglia lysosomal enzyme activity, could prevent microglia from causing neuronal injury. Hence, in addition to the numerous novel proteins that are potentially important in microglial activation-mediated neurodegeneration revealed by the search, the study has indicated that the early release of lysosomal enzymes in microglial cells would contribute to LPS-activated inflammatory response.