A large amount of clinical evidence has revealed that ketamine can relieve fentanyl-induced hyperalgesia. However, the underlying mechanism is still unclear. In the current study, a single dose of ketamine (5 mg/kg or 10 mg/kg), TAK-242 (3 mg/kg), or saline was intraperitoneally injected into rats 15 min before four subcutaneous injections of fentanyl. Results revealed that pre-administration of ketamine alleviated fentanyl-induced hyperalgesia according to hind paw-pressure and paw withdrawal tests. High-dose ketamine can reverse the expression of toll-like receptor-dimer (d-TLR4), phospho-nuclear factor kappa-B (p-NF-KB,p-p65), cyclooxygenase-2 (COX-2), interleukin-1(3 (IL-1(3), and tumor necrosis factor-a (TNF-a) 1 d after fentanyl injection in the spinal cord. Moreover, fentanylinduced-hyperalgesia and changes in the expression of the aforementioned proteins can be attenuated by TAK-242, an inhibitor of TLR4, as well as ketamine. Importantly, TLR4, p-p65, COX-2, and IL 1(3 were expressed in neurons but not in glial cells in the spinal cord 1 d after fentanyl injection. In conclusion, results suggested that a single dose of ketamine can relieve fentanyl-induced-hyperalgesia via the TLR4/NF-KB pathway in spinal cord neurons.