<b><i>Background/Aims:</i></b> Guanidinoacetic acid (GAA) is an experimental dietary additive and has been reported to induce methyl depletion when provided by the diet. However, no study evaluated whether supplemental GAA affects DNA methylation, a critical epigenetic process for genome regulation. <b><i>Methods:</i></b> In this open-label, repeated-measure interventional trial, we evaluated the impact of 12 weeks of GAA supplementation on global DNA methylation in 14 healthy participants (8 women and 6 men, age 22.2 ± 2.3 years, body mass index 24.8 ± 5.7). <b><i>Results:</i></b> Dietary provision of GAA had no effect on global DNA methylation, with 5-methylcytosine (m⁵C) nonsignificantly increased by 13.4% at postadministration when averaged across participants (95% confidence interval –5.5 to 32.3; <i>p</i> = 0.26). Notable DNA hypomethylation (corresponding to a 5% drop in m⁵C) was found in 3 of 14 participants at follow-up. <b><i>Conclusion:</i></b> Global DNA methylation seems to be unaltered by dietary provision of 3 g of GAA per day for 12 weeks in healthy men and women.