Background: Low-dose methotrexate (MTX) is the first-line therapy in early rheumatoid arthritis (eRA). Up to 40% of eRA patients do not benefit from MTX therapy. MTX has been shown to inhibit one-carbon metabolism, which is involved in the donation of methyl groups. In this study, we investigate baseline global DNA methylation and changes in DNA methylation during treatment in relation to clinical non-response after 3 months of MTX treatment. Methods: Two hundred ninety-four blood samples were collected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, ISRCTN26791028), a multicenter, stratified single-blind clinical trial of eRA patients. Global DNA (hydroxy)methylation was quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and validated with a global DNA LINE-1 methylation technique. MTX response was determined as ΔDAS28. Additionally, patients were stratified into two response groups according to the European League Against Rheumatism (EULAR) response criteria. Associations between global DNA methylation and response were examined using univariate regression models adjusted for baseline DAS28, baseline erythrocyte folate levels, and body mass index (BMI). Results: Higher baseline global DNA methylation was associated with less decrease of DAS28 (β = 0.15, p = 0.013) and with MTX non-response (OR = 0.010, 95% CI = 0.001-0.188). This result was validated in LINE-1 elements (β = 0.22, p = 0.026). Changes in global DNA (hydroxy)methylation were not associated with MTX response over 3 months. Conclusions: These results show that higher baseline global DNA methylation in treatment naïve eRA patients is associated with decreased clinical response after 3 months of treatment of eRA patients and can be further evaluated as a predictor for MTX therapy non-response. Trial registration: ISRCTN, ISRCTN26791028, registered 23 August 2007 - retrospectively registered.