Piwi-Interacting RNA (piRNA) is the largest class of small noncoding RNA and is 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, the overexpression of spinal piR-DQ541777 in naive 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 cdk5rap1 promoter and consequently reduced the expression of Cdk5rap1, which was reversed by the knockdown of piR-DQ541777 and mimicked by the overexpression of piR-DQ541777 in naive mice. Finally, piR-DQ541777 increased the methylation level of CpG islands by recruiting DNA methyltransferase 3A (DNMT3a) to cdk5rap1 promoter. In conclusion, this study represents a novel role of piR-DQ541777 in the regulation of neuropathic pain through the methylation of cdk5rap1.