DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.