As a therapeutic target for neuropathic pain, the anti-nociceptive effects of α 2-adrenoceptors (α2AR) have attracted attention. Dexmedetomidine (DEX), a potent and highly selective α2AR agonist, has exhibited significant analgesic effects in neuropathic pain, but the underlying mechanism has remained elusive. The present study investigated the effect of DEX on Toll-like receptor (TLR)4 and nuclear factor (NF)-κB p65 expression, as well as the production of pro-inflammatory cytokines. The rat monoarthritis (MA) model was induced by intra-articular injection of complete Freund's adjuvant (CFA) at the ankle joint. After induction of MA, the rats were intrathecally treated with normal saline or DEX (2.5 µg) for 3 consecutive days. The concentration of interleukin-1β and -6 as well as tumor necrosis factor-α was examined by ELISA. The expression levels of TLR4 and NF-κB p65 were determined by western blot analysis and immunohistochemistry. The results indicated that the pro-inflammatory cytokines TLR4 and NF-κB p65 were significantly upregulated in MA rats. DEX treatment markedly reduced mechanical and thermal hyperalgesia, suppressed MA-induced elevation of the pro-inflammatory cytokines and inhibited the TLR4/NF-κB p65 pathway, while these effects were blocked by pre-treatment with the selective α2AR antagonist BRL44408 (15 µg) at 30 min prior to CFA injection. These results suggested that DEX has an anti-nociceptive effect via suppressing the TLR4/NF-κB p65 pathway.