Abstract: DNA methylation is the first epigenetic modification found in humans. Abnormal changes in DNA methylation are closely associated with the development and progression of diseases. Acupuncture, an important component of traditional Chinese medicine, has been shown to have significant therapeutic efficacy. The mechanisms underlying acupuncture are complex, involving physiological and pathological processes of integrated interactions across multiple targets. However, current research mostly focuses on a single target, highlighting the need for a more upstream approach to the investigation of the mechanisms. Herein, we reviewed studies on the direct or indirect regulation of DNA methylation via acupuncture. We also discussed its mechanisms of action in pain, obesity, depression, and Alzheimer disease, in order to provide a new perspective on the therapeutic mechanisms of acupuncture and the role of DNA methylation in the field of acupuncture research. Future research should concentrate on the effect of acupuncture on the DNA methylation of specific genes, the quantification of changes in DNA methylation at different acupoints, the development of individualized acupuncture prescriptions, further investigation of the specificity of the effects at different acupoint, and the expansion of the research to integrate epigenetics and genomics. This will provide a theoretical basis for the internationalization and the promotion of clinical application of acupuncture.