MicroRNA are emerging as significant regulators of neuropathic pain progression. In addition, neuroinflammation contributes a lot to neuropathic pain. miR-26a-5p has been identified as an inflammation-associated miRNA in multiple pathological processes. However, little is known about the biological role of miR-26a-5p in neuroinflammation and neuropathic pain development. Therefore, we aimed to investigate the function of miR-26a-5p in neuropathic pain by establishing a rat model using chronic sciatic nerve injury (CCI). A significant decrease of miR-26a-5p expression was observed in the spinal cord tissues form the CCI rats compared to the control group. Moreover, overexpression of miR-26a-5p significantly repressed neuropathic pain and neuroinflammation in CCI rats. MAPK6 was identified as a direct downstream target gene of miR-26a-5p and confirmed by dual-luciferase reporter assays. As displayed, overexpression of miR-26a-5p greatly reduced MAPK6 levels in vitro and in vivo. Meanwhile, MAPK6 expression and miR-26a-5p were oppositely correlated in CCI rats. Furthermore, up-regulation of MAPK6 obviously reversed the suppressive effect of miR-26a-5p on neuroinflammation and neuropathic pain progression. Taken these together, our results implied that miR-26a-5p could act as a negative regulator of neuropathic pain development through targeting MAPK6, which indicated that miR-26a-5p might serve as a potential therapeutic target for neuropathic pain.