Astrocytes activation has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including bacterial infections, cerebral ischemia, and Parkinson's diseases. Acetylpuerarin is a newly modified isoflavone based on puerarin that has neuroprotective and antioxidant effects. In this study, we investigated the anti-inflammatory action of acetylpuerarin in regulating the eicosanoids generation and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced production of arachidonic acid (AA) metabolites in primary rat astrocytes. The results showed that acetylpuerarin concentration dependently inhibited the LPS-induced production of AA metabolites such as prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), and acetylpuerarin significantly attenuated the expression and immunoreactivity of group V secretory phospholipase A2 (sPLA2) protein induced by LPS in astrocytes. Furthermore, in astrocytes pretreated with acetylpuerarin, the time course of phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and of cytosolic PLA2 alpha (cPLA2α) and expression of transcription factors, nuclear factor kappa B (NF-κB), was markedly truncated. Acetylpuerarin concentration dependently abolished the LPS-induced expressions of AA-metabolizing enzymes including cyclooxygenase-2 (COX-2) and lipooxygenase-5 (LOX-5). This study indicates that acetylpuerarin inhibited LPS-induced AA-metabolizing enzymes and AA metabolites in astrocytes via downregulation expression of group V sPLA2 and phosphorylation of ERK1/2, cPLA2α, and NF-κB. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of acetylpuerarin.