Chronic neuropathic pain is a common symptom in clinical practice and patients with chronic pain are subject to a greatly impaired quality of life. Grafted genetically-modified cells secreting enkephalin have been considered an encouraging treatment for chronic pain. Importantly, the transplanted cell as a therapeutic agent should be reproducible, safe, and controllable. In this study, by combining a tetracycline-controlled (Tet-on) gene expression system and immortalized astrocytes, we attempted to engineer an immortalized astrocyte line carrying the human preproenkephalin gene (IASL/hPPE) under the transcriptional control of doxycycline. These cells were then implanted into the subarachnoid space of chronic constrictive injury (CCI) rats and their analgesic potential was investigated by behavioral tests. The results showed that the secretion of enkephalin from IASL/hPPE cells could be switched on and off under the regulation of doxycycline in a dose-dependent manner. In addition, the mechanical and thermal hyperalgesia induced by CCI was significantly alleviated during the 2-7 week period after grafts of IASL/hPPE cells and the analgesic effect could be regulated by doxycycline. Moreover, spinal enkephalin level could be modulated by the presence or absence of doxycycline in drinking water. Taken together, these data suggest that regulatable release of enkephalin from transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain. Although improvements in the Tet-on system are necessary, this may provide an alternative approach for ex vivo cell transplantation to treat chronic pain.