In response to brain insults, microglia, the resident inflammatory cells in CNS, migrate into injured sites to initiate inflammatory responses in brain. ATP, released from apoptotic or necrotic cells induce chemoattractive responses but the mechanism is not clear yet. In this study, we investigated whether ATP modulates microglial migration by regulating the activity of matrix metalloproteinases (MMPs). ATP induced rapid microglial migration and increased the activity of MMP-9 in the culture supernatants (secreted compartments) in a concentrationdependent manner. The increased activity of secreted MMP-9 is due to the increased protein secretion, but not by the increased MMP-9 mRNA and protein expression. Inhibition of MMP-9 activity by treatment with specific inhibitors including GM6001 and SB-3CT prevented ATPinduced microglial migration. ATP-induced microglial migration was also inhibited by P2Y receptor antagonists including clopidogrel as well as PI3K inhibitor such as wortmanin. Taken together, ATP non-transcriptionally increased MMP-9 activity by activation of P2Y and PI3K. The results from the present investigation may provide further insights into the regulation of the activity of MMP-9 during microglial migration, which may play essential role in the regulation of inflammatory responses in pathological situations such as neurodegenerative disorders.