Piwi-interacting RNA (piRNA) is the largest class of small noncoding RNA and involved in various physiological and pathological processes. However, whether it has a role in pain modulation remains unknown. In the present study, we found that spinal piRNA-DQ541777 (piR-DQ541777) was significantly increased in the male mouse model of sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain. Knockdown of spinal piR-DQ541777 alleviated CCI-induced thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization. However, overexpression of spinal piR-DQ541777 in naïve mice produced pain behaviors and increased spinal neuron sensitization. Furthermore, we found that piR-DQ541777 regulates pain behaviors by targeting CDK5 regulatory subunit-associated protein 1 (Cdk5rap1). CCI increased the methylation level of CpG islands in the promoter and consequent reduced the expression of Cdk5rap1, which was reversed by knockdown of piR-DQ541777 and mimicked by overexpression of piR-DQ541777 in naïve mice. Finally, piR-DQ541777 increased the methylation level of CpG islands by recruiting DNA methyltransferase 3A (DNMT3a) to promoter. In conclusion, this study represents a novel role of piR-DQ541777 in the regulation of neuropathic pain through methylation of Chronic pain affects approximately 20% of the world's population and is Although we have studied the neurobiological mechanism of neuropathic pain for decades, there is still no ideal drug available to treat it. This work that a novel role of piR-DQ541777 in the regulation of neuropathic pain through methylation of Our findings provide the first evidence of the regulatory effect of piRNAs on neuropathic pain, which may improve our understanding of pain mechanisms and lead to the discovery of novel drug targets for the prevention and treatment of neuropathic pain.