Systemic lupus erythematosus (SLE) is a chronic, potentially fatal systemic autoimmune disease characterized by the production of autoantibodies against a wide range of self-antigens. To investigate the role of the 5-hmC DNA modification with regard to the onset of SLE, we compared the levels 5-hmC between SLE patients and normal controls. Whole blood was obtained from patients, and genomic DNA was extracted. Using the hMeDIP-chip analysis and validation by quantitative RT-PCR (RT-qPCR), we identified the differentially hydroxymethylated regions that are associated with SLE. There were 1,701 genes with significantly different 5-hmC levels at the promoter region in the SLE patients compared with the normal controls. The CpG islands of 3,826 genes showed significantly different 5-hmC levels in the SLE patients compared with the normal controls. Out of the differentially hydroxymethylated genes, three were selected for validation, including TREX1, CDKN1A and CDKN1B. The hydroxymethylation levels of the three genes were confirmed by RT-qPCR. The results suggested that there were significant alterations of 5-hmC in SLE patients. Thus, these differentially hydroxymethylated genes may contribute to the pathogenesis of SLE. These findings show the significance of 5-hmC as a potential biomarker or promising target for epigenetic-based SLE therapies.