Several drugs and agents are currently used for the treatment of neuropathic pain. Among them amitriptyline, a tricyclic antidepressant drug, represent a first line treatment. Despite its well-documented clinical efficacy, amitriptyline is ineffective in some animal models of neuropathic pain. The aim of this study was to investigate into amitriptyline poor efficacy in neuropathic pain and to determine the role of c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the analgesic effects of this drug. Experiments were performed in mice with painful peripheral neuropathies due to the antiretroviral agent 2,3-dideoxycytidine (ddC), and with the partial sciatic nerve injury produced in the spared nerve injury model (SNI). In mice subjected to SNI and antiretroviral treatment, amitriptyline did not attenuate mechanical allodynia and thermal hyperalgesia. Conversely, intrathecal injection of the JNK inhibitor SP600125 prevented SNI and ddC-induced nociceptive behavior and, its inactive dose co-administrated with amitriptyline induced an antinociceptive effect. Western blotting analysis showed an upregulation of p-JNK in the lumbar spinal cord of SNI and ddC-exposed mice, that was further enhanced after amitriptyline administration. Additionally, amitriptyline further promoted astrocyte activation in neuropathic mice, as illustrated by the increased expression of glial fibrillary acidic protein (GFAP), that was attenuated by intrathecal injection of the JNK inhibitor. These data indicate astrocyte JNK activation as counteracting pathway to amitriptyline analgesic response. Targeting the JNK pathway in spinal astroglia may present an efficient way to improve the analgesic efficacy of amitriptyline in the neuropathic pain treatment.