MOTS-c, a recently discovered mitochondrial-derived peptide, plays an important role in many physiological and pathological functions adenosine monophosphate-activated protein kinase (AMPK) activation. Numerous studies have demonstrated that AMPK is an emerging target for the modulation of neuropathic pain. Meanwhile, microglia-activation-evoked neuroinflammation is known to contribute to the development and progression of neuropathic pain. MOTS-c is also known to inhibit microglia activation, chemokine and cytokine expression, and innate immune responses. Accordingly, in this study, we evaluated the effects of MOTS-c on neuropathic pain and investigated the putative underlying mechanisms. We found that MOTS-c levels in plasma and spinal dorsal horn were significantly lower in mice with spared nerve injury (SNI)-induced neuropathic pain than in control animals. Accordingly, MOTS-c treatment produced pronounced dose-dependent antinociceptive effects in SNI mice; however, these effects were blocked by dorsomorphin, an AMPK inhibitor, but not naloxone, a nonselective opioid receptor antagonist. Moreover, intrathecal (i.t.) injection of MOTS-c significantly enhanced AMPKα phosphorylation in the lumbar spinal cord of SNI mice. MOTS-c also significantly inhibited proinflammatory cytokine production and microglia activation in the spinal cord. The antinociceptive effects of MOTS-c were retained even when microglia activation in the spinal cord was inhibited by minocycline pretreatment, indicating that spinal cord microglia are dispensable for the antiallodynic effects of MOTS-c. In the spinal dorsal horn, MOTS-c treatment inhibited c-Fos expression and oxidative damage mainly in neurons rather than microglia. Finally, in contrast to morphine, i.t. administration of MOTS-c resulted in limited side effects relating to antinociceptive tolerance, gastrointestinal transit inhibition, locomotor function, and motor coordination. Collectively, the present study is the first to provide evidence that MOTS-c may be a promising therapeutic target for neuropathic pain.