Background: Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS). Methodology/Principal Findings: BV-2 cells were pretreated with gastrodin (30, 40, and 60 mu M) for 1 h and then stimulated with LPS (1 mu g/ml) for another 4 h. The effects on proinflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), are analysed by double-immunofluorescence labeling and RT-PCR assay. To reveal the mechanisms of action of gastrodin we investigated the involvement of mitogen-activated protein kinases (MAPKs) cascades and their downstream transcription factors, nuclear factor-kappa B (NF-kappa B) and cyclic AMP-responsive element (CRE)-binding protein (CREB). Gastrodin significantly reduced the LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-alpha, IL-1 beta and NF-kappa B. LPS (1 mu g/ml, 30 min)-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) and this was inhibited by pretreatment of BV-2 cells with different concentrations of gastrodin (30, 40, and 60 mu M). In addition, gastrodin blocked LPS-induced phosphorylation of inhibitor kappa B-alpha (I kappa B-alpha) (and hence the activation of NF-kappa B) and of CREB, respectively. Conclusion and Implications: This study indicates that gastrodin significantly attenuate levels of neurotoxic proinflammatory mediators and proinflammatory cytokines by inhibition of the NF-kappa B signaling pathway and phosphorylation of MAPKs in LPS-stimulated microglial cells. Arising from the above, we suggest that gastrodin has a potential as an anti-inflammatory drug candidate in neurodegenerative diseases.