Background <p>Regular exercise promotes health through multiple mechanisms, with DNA methylation serving as a key epigenetic modification involved in the molecular regulation induced by exercise. Peripheral blood, as an accessible tissue, is commonly used to study exercise-induced DNA methylation changes.</p> Objective <p>This systematic review aims to synthesize evidence from human exercise intervention studies on the effects of exercise on peripheral blood DNA methylation and related epigenetic markers, and to evaluate their potential as biomarkers of exercise efficacy.</p> Methods <p>The study protocol was registered in PROSPERO (CRD420251069398). A systematic search was conducted in Pubmed, Web of Science, Cochrane Library, Embase and Scopus databases from database inception through 29 May 2025 for all English-language studies. Eligible studies were included. Risk of bias of randomized controlled trials were assessed using the Cochrane collaboration’s tool (version 5.1.0) and quasi-experimental studies were assessed using the JBI Critical Appraisal Checklists for Quasi-Experimental Studies. The results were summarized using narrative synthesis.</p> Results <p>Twenty-nine studies were included, covering both acute and long-term exercise interventions. Acute exercise induced minor and mostly statistically nonsignificant changes in peripheral blood DNA methylation, whereas long-term exercise elicited significant methylation remodeling in genes related to metabolism, inflammation, and immune function. Exercise dose (frequency, intensity, and duration) and population characteristics influenced the magnitude and scope of methylation responses.</p> Conclusions <p>Peripheral blood DNA methylation provides robust evidence for exercise-mediated systemic epigenetic regulation and shows promise as a biomarker for exercise adaptation and intervention effects. Future research should optimize intervention designs and methodological standards to deepen understanding of exercise epigenetic mechanisms.</p>