Spinal cord injury (SCI), characterized by sensory disturbance and motor deficits, is associated with excessive inflammatory cytokine production of microglial cells. Previous studies have demonstrated High mobility group box 2 (HMGB2) as a microglial pro-inflammatory factor in stroke. This present study aims to evaluate the function of HMGB2 in a SCI rat model induced by striking the spinal cord at T9 to T12 using a rod. Our results showed that the levels of HMGB2 were significantly increased in the spinal cord tissues of SCI rats. Besides, HMGB2 downregulation was achieved by receiving an injection of lentivirus encoding HMGB2 shRNA in the spinal cord. Knockdown of HMGB2 suppressed SCI-induced microglial activation and neuroinflammation, as well as alleviated neuronal loss. In addition, we confirmed that HMGB2 silencing lessened lipopolysaccharide (LPS)-induced neuroinflammation in BV-2 cells. Furthermore, our findings demonstrated that HMGB2 knockdown suppressed the canonical nuclear factor of kB (NF-κB) signaling pathway both in vivo and in vitro. Collectively, this study manifested strong anti-inflammatory roles of HMGB2 knockdown on microglia-mediated neuroinflammation and suggested that HMGB2 might serve as a potential target for SCI therapy.