Objective To investigate the effects of spinal cannabinoid type 2 receptor (CB₂R) and microglia activation on hyperalgesia in neuropathic pain mice. Methods Male C57/BL mice were randomly divided into six groups:sham, spinal nerve ligation (SNL), SNL+CB₂R agonist AM1241 (SNL+AM1241), SNL+microglia inhibitor minocycline (SNL+ minocycline), SNL+small interfering RNA (siRNA) targeting CB₂R (SNL+siRNA), and SNL+siRNA+minocycline groups. A neuropathic pain mouse model was established by SNL. The expression levels of spinal CB₂R and microglia-specific protein ionized calcium-binding adapter molecule 1 (IBA-1) were determined by Western blotting, mechanical pain thresholds were measured by Von Frey, spinal microglia activation was observed by IBA-1 immunofluorescence, and the expression levels of inflammatory factors in spinal cord dialysate were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Electrophysiology was applied to observe the effect of CB₂R agonist on spontaneous inhibitory postsynaptic current (sIPSC) in the spinal dorsal horn. Results Compared with the sham group, the expression of CB₂R in spinal cord was significantly decreased in the SNL group (P<0.012 5), the pain threshold was significantly reduced (P<0.016 7), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P<0.008 3), and the mRNA expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were significantly increased (all P<0.008 3). After intrathecal injection of CB₂R agonist AM1241 or microglial inhibitor minocycline, compared with the SNL group, the pain thresholds of mice were significantly increased in the SNL+AM1241 and SNL+minocycline groups (both P<0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly decreased (both P<0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly decreased (all P<0.008 3). After targeted interfering CB₂R expression by siRNA, compared with the SNL group, the pain threshold was significantly decreased in the SNL+siRNA group (P<0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P<0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly increased (all P<0.008 3); while intrathecal injection of minocycline significantly reversed the above changes (all P<0.008 3). Intervention in vitro of AM1241 could significantly enhance the frequency and amplitude of sIPSC in the spinal dorsal horn (both P<0.05), while continuous treatment with minocycline inhibited the enhancement effects of AM1241 on sIPSC. Conclusion CB₂R can reduce the neuroinflammatory responses and enhance the inhibitory electrical activity in the spinal cord by inhibiting spinal microglia activation, thereby alleviating hyperalgesia of neuropathic pain in mice.