Abstract Background: Treatment of uremia is now dominated by dialysis; in some cases, patients are treated with dialysis for decades, but overall outcomes are disappointing. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of uremia, but the specific biomarkers of uremia have not been fully elucidated. To date, our knowledge about the alterations in DNA 5-hydroxymethylcytosine (5-hmC) in uremia is unclear, to investigate the role of DNA 5-hmC in the onset of uremia, we performed hMeDIP-chip between the uremia patients and the normal controls from the experiment to identify differentially expressed 5-hmC in uremia-associated samples. Methods: Extract genomic DNA, using hMeDIP-chip technology of Active Motif companies for the analysis of genome-wide DNA 5-hmC, and quantitative real-time PCR confirmation to identify differentially expressed 5-hmC level in uremia-associated samples. Results: There were 1875 genes in gene Promoter, which displayed significant 5-hmC differences in uremia patients compared with normal controls. Among these genes, 960 genes displayed increased 5-hmC and 915 genes decreased 5-hmC. 4063 genes in CpG Islands displayed significant 5-hmC differences in uremia patients compared with normal controls. Among these genes, 1780 genes displayed increased 5-hmC and 2283 genes decreased 5-hmC. Three positive genes, HMGCR, THBD, and STAT3 were confirmed by quantitative real-time PCR. Conclusion: Our studies indicate the significant alterations of 5-hmC. There is a correlation of gene modification 5-hmC in uremia patients. Such novel findings show the significance of 5-hmC as a potential biomarker or promising target for epigenetic-based uremia therapies.