Nitric oxide (NO) may act as a neurotransmitter or neuromodulator that contributes to chronic pain conditions. The neuronal nitric oxide synthase (nNOS) is a key enzyme for NO production in neuronal tissues, but the effects of systemic or spinal treatment with specific or non-specific nNOS inhibitors on neuropathic pain in the rats are conflicting. Here, we combined an enzyme gene knock out strategy with a pharmacologic approach to study the role of nNOS in neuropathic pain induced by L5 spinal nerve injury in the mice. The nNOS knockout mice displayed impaired thermal and mechanical pain hypersensitivities during the development and maintenance of neuropathic pain. Intraperitoneal administration of L-NG-nitro-arginine methyl ester (L-NAME, 100 mg/kg), a non-selective NOS inhibitor, significantly blocked mechanical pain hypersensitivity in wild type mice at day 7 post-nerve injury. The inactive form D- NG-nitro-arginine methyl ester (D-NAME, 100 mg/kg) had no effect. Furthermore, intrathecal injection of L-NAME (30 µg/10 µl), but not D-NAME (30 µg/10 µl), also markedly attenuated mechanical pain hypersensitivity in the wild type mice at day 7 post-nerve injury. Our findings suggest that NOS, particularly spinal cord nNOS, might be activated after nerve injury and contribute to the central mechanism of neuropathic pain in the mice.