BACKGROUND: The development of Crohn's disease (CD) involves immune cell signaling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4 + lymphocytes has not been evaluated. MATERIALS AND METHODS: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4 + cells from 21 CD patients and 12 age and sex matched controls. Data was analyzed for differentially methylated CpGs (DMCs) and methylated regions (DMRs). Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin (by ATAC-seq) and CCCTC-binding factor (CTCF) binding sites (by ChIP-seq) between peripherally-derived Th17 and Treg cells. RESULTS: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119,051 DMCs and 8,113 DMRs were detected. While hyper-methylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signaling pathway. The differentially enriched ATAC regions in Th17 cells (compared to Tregs) were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites. CONCLUSIONS: The methylome of CD patients demonstrate an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4 + cells.