Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease of unknown origin, which exhibits a complex heterogeneity in its pathophysiological background, resulting in differential responses to a range of therapies and poor long-term prognosis. RA synovial fibroblasts (RASFs) are key player cells in RA pathogenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the process of diagnosis and prognosis of various human diseases. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with a high resolution CpG microarray for discovery of novel hypermethylated genes in RASFs. Thirteen genes (APEX1, EBF3, EGR2, EN1, IRX1, IRX6, KIF12, LHX2, MIPOL1, SGTA, SIN3A, TOLLIP, and ZHX2) with three consecutive hypermethylated probes were isolated as candidate genes through two CpG microarrays. Pyrosequencing assay was performed to validate the methylation status of TGF-β signaling components, EBF3 and IRX1 genes in RASFs and osteoarthritis (OA) SFs. Hypermethylation at CpG sites in the EBF3 and IRX1 genes was observed with a high methylation index (MI) in RASFs (52.5% and 41.4%, respectively), while a lower MI was observ ed in OASFs and h ealthy SFs (13.2% for EBF3 and 4.3% for IRX1). In addition, RT-PCR analysis showed a remarkable decrease in their mRNA expression in the RA group, compared with the OA or healthy control, and their reduction levels correlated with MI. The current findings suggest that methylation-associated down-regulation of EBF3 and IRX1 genes may play an important role in a pathogenic effect of TGF-β on RASFs. However, further clinical validation with large numbers of patients is needed in order to confirm our findings.