Microglial activation plays an important role in neroinflammation following ischemic stroke. Activated microglial cells can then migrate to the site of injury to proliferate and release substances which induce secondary brain damage. It has been shown that microglial migration is associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathways. The Chinese formula, GuaLou GuiZhi decoction (GLGZD), has long been administered in clinical practice for the treatment of post-stroke disabilities, such as muscular spasticity. In a previous study, we demonstrated that the anti-inflammtory effects of GLGZD were mediated by the TLR4/NF-κB pathway in lipopolysaccharide (LPS)-stimulated microglial cells. Therefore, in this study, we evaluated the role of GLGZD in microglial migration by performing scratch wound assays and migration assays. We wished to elucidate the cellular and molecular mechanisms elicited by this TCM formula in microglial-induced inflammation by evaluating the release and expression of chemotactic cytokines [monocyte chemo-attractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and interleukin (IL)-8] by ELISA and quantitative PCR. Our results revealed that the migration of microglial cells was enhanced in the presence of LPS (100 ng/ml); however, GLGZD (100 µg/ml) significantly inhibited cell motility and the production of chemokines through the inhibition of the activation of the p38 and c-Jun N-terminal protein kinase (JNK) signaling pathway. We demonstrate the potential of GLGZD in the modulation of microglial motility by investigating the effects of GLGZD on microglial migration induced by LPS. Taken together, our data suggest that GLGZD per se cannot trigger microglial motility, whereas GLGZD impedes LPS-induced microglial migration through the activation of the MAPK signaling pathway. These results provide further evidence of the anti-inflammatory effects of GLGZD and its potential for use in the treatment of ischemic stroke.