In the current study, we reveal that in astrocytes the VIB Ca(2+)-independent phospholipase A(2) is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2), docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca(2+)-independent phospholipase A(2) significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca(2+)-independent phospholipase A(2) negatively controls prostaglandin production.