Glial cells have various important roles in regulation of brain functions. For such events, extracellular nucleotides/P2 receptors have central roles. Although there have been huge amount of literature about activation of P2 receptors and glial functions, little is known about what happens in glia or the brain if glial P2 receptor is inhibited. Here we show that the inhibition of P2 receptors in astrocytes, the most abundant glial cells and cause a constitutive release of nucleotides, resulted in secretion of metalloproteinase-9 (MMP-9), a metal-dependent endopeptidase that degrades extracellular matrix molecules and is important in regulation of brain remodeling. When cultured astrocytes were treated with apyrase (ecto-nucleotidase), reactive blue 2 (P2 receptor antagonist), and pertussis toxin, they secreted MMP-9, suggesting that Gi-coupled P2Y receptor-mediated signals constitutively suppress the production of MMP-9. Among Gi-coupled P2Y receptors, we found that an inhibition of P2Y(14) receptor, a receptor for nucleotide-sugars such as UDP-glucose, is responsible for the production of MMP-9 by pharmacological and molecular biochemical analysis. As for the mechanisms, the inhibition of P2Y(14) receptors resulted in the release of tumor necrosis factor (TNF)-α which then acted on astrocytes to induce MMP-9. Taken together, our results suggest that the constitutive releases of nucleotide-sugars in astrocytes should play an important role in maintaining the normal status of the cell, through Gi-coupled P2Y(14) receptors, and when the signal is removed, the cells start to release TNF-α, which then acts on astrocytes in a feedback fashion to boost MMP-9 synthesis and secretion.