The cytochrome P450 proteins (CYP450s) have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases. CYP26A1, a member of the CYP450 family, carries out the oxidative metabolism of retinoic acid (RA), the active metabolite of vitamin A. Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation (SNL). CYP26A1 was mainly expressed in spinal neurons and astrocytes. HPLC analysis displayed that the content of all-trans-RA (at-RA), the substrate of CYP26A1, was reduced in the spinal cord on day 7 after SNL. Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain. Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine (IL-10) production. The RA receptors RARα, RXRβ, and RXRγ were expressed in spinal neurons and glial cells. The promoter of Il-10 has several binding sites for RA receptors, and at-RA directly increased Il-10 mRNA expression in vitro. Finally, intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia. Collectively, the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation. CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.