Objective：To investigate the role of Toll-like receptor（TLR）/NF-κB signaling pathway in methamphetamine（METH）-induced primary microglial activation and inflammatory reaction. Methods：Primary microglial cells were isolated and cultured，then the purity of primary microglia was detected by immunofluorescence. Cells were treated with METH（300 μmol/L） for 0 min，15 min，30 min，1 h，3 h，6 h，12 h，and 24 h，and the activations of primary microglia and NF-κB pathway were detected by Western blot. The inflammatory factors（TNF-α，IL-1β，and IL-6）and TLR1-9，11 mRNA levels were detected by real-time PCR. Results：The purity of primary microglial cells was above 95%. After exposure to METH，the primary microglia activation marker IBA-1 was increased and the NF-κB pathway was activated. In addition，the mRNA levels of inflammatory factors（TNF-α，IL-1β，IL-6） and TLR2，4-5，7-9，11 were significantly increased（P < 0.05），while the level of TLR1 mRNA was significantly reduced. Conclusion：METH can activate primary microglial cells and promote the release of inflammatory factors by regulating the TLR/NF-κB signaling pathway. Therefore，TLR can be potential targets for METH neuritis response intervention with certain therapeutic significance.