Glial glutamate transporter-1 (GLT-1) plays an essential role in the maintenance of glutamate homeostasis and is involved in the development and maintenance of pathological pain. The present study was undertaken (1) to observe the anti-nociceptive effects of ceftriaxone (Cef) in a chronic neuropathic pain model induced by chronic constrictive nerve injury (CCI) of the sciatic nerve and (2) to identify the role of spinal GLT-1 in the process. CCI induced significant thermal hyperalgesia and mechanical allodynia, which began from postoperative day 3 and lasted to day 21. This long-term hyperalgesia was accompanied by significant down-regulation of GLT-1 expression in the L4-L6 segments of the spinal dorsal horn, as revealed by immunohistochemistry and Western blot. Intraperitoneal preventive and therapeutic administration of Cef effectively prevented or reversed, respectively, the development of thermal hyperalgesia, mechanical allodynia, and GLT-1 down-regulation in the spinal dorsal horn. To further determine whether the above anti-nociceptive effects of Cef are a result of the up-regulation of spinal GLT-1 expression and its function, we further observed the effects of intrathecal administration of Cef in the same model. It was found that intrathecal administration of Cef led to the specific up-regulation of GLT-1 expression and glutamate uptake ((3)H-glutamate) in the spinal dorsal horn, and similar anti-nociceptive effects to those of intraperitoneal administration of Cef. The above effects of intrathecal Cef administration were all significantly inhibited by intrathecal administration of GLT-1 antisense oligodeoxynucleotides (As-ODNs). These results indicate that Cef plays an anti-nociceptive role by up-regulating spinal GLT-1 expression and its function.