Neuropathic pain is one of most intense types of chronic pain. Numerous studies investigating neuropathic pain have described the critical involvement of microglia in the spinal cord. Previous studies have indicated that activation of large conductance Ca2+‑activated K+ (BK) channels contributes to microglial activation in the spinal dorsal horn (SDH) and the generation of neuropathic pain. However, the specific role of BK channels in spinal microglia in neuropathic pain has not been fully addressed in previous studies, as BK channel inhibitors were used to inhibit microglial BK channel based on their inhibitory kinetics. We previously identified that Ca2+‑activated K+ channel β3 auxiliary subunit (KCNMB3), which is an auxiliary subunit of BK channels and regulates gating properties of the channel, is exclusively expressed in microglia in the spinal cord. The present study analyzed the role of BK channels in spinal microglia in neuropathic pain using a spinal microglia‑specific BK channel knockdown method, with intrathecal injection of KCNMB3 small interfering RNA. Neuropathic pain was significantly attenuated in KCNMB3 knockdown mice. Increases in the number of microglia in the SDH following nerve injury were attenuated by KCNMB3 knockdown. Furthermore, increased levels of pain‑associated molecules in the SDH were attenuated in KCNMB3 knockdown mice. Attempts were also made to analyze the effects of KCNMB3 knockdown on chronic pain. KCNMB3 knockdown ameliorated chronic pain and inhibited the expression levels of pain‑associated molecules in the SDH. The results from the present study suggested that BK channels modulated the activation state of spinal microglia, and that KCNMB3 is a potential therapeutic target for neuropathic pain.