Abstract Body (Do not enter title and authors here): Valvular interstitial cells (VICs), the valves’ predominant cell type, are crucial in ensuring proper valve function, structural integrity, tissue repair and valve homeostasis. DNA methylation regulates gene expression profiles and a matched aortic to mitral human VIC methylation analysis will further our understanding of aortic- and mitral-specific VIC biology and pathobiology. This is the first study analyzing methylation profiles of non-diseased, matched human aortic and mitral VICs. We analyzed the differential methylation fingerprint of 12 non-diseased aortic and mitral VICs (10 males:2 females; 42–64 years) extracted from de-endothelialized leaflets using reduced representation bisulfite sequencing (RRBS) on HiSeq2500. Analysis of methylation levels of 12,676 genome-wide promoters revealed 651 differentially methylated (DM) promoters. The top 3 DM promoters were linked to TBX4 (meth diff = -28.9%, P = 5.2E-135) essential for valve morphogenesis, to EXOC3L2 (meth diff = 25.7%, P = 1.3E-92) and NT5DC2 (meth diff = -20.3%, P = 7.1E-69) potentially implicated in ECM remodeling, the former via vesicle trafficking and the latter through nucleotide metabolism. Functional classification and network analysis showed that the genes associated with the DM promoters were enriched for WNT-, TGFβ-, FGF-, EGF- and PDGF- pathways involved in physiological processes such as valve development, repair and VIC regulation, as well as pathological processes such as calcification. Additional enrichment was detected for integrin- and cadherin- pathways involved in cell-cell-ECM interactions and endothelin- and VEGF pathways involved in hemodynamic regulation. This work provides the first insight into the differential methylation regulation patterns of human aortic and mitral VICs. Understanding how aortic and mitral VICs are differentially epigenetically regulated will extend our understanding of their role in aortic- and mitral-specific leaflet homeostasis, susceptibility to disease processes and will contribute to dynamic innovative experimental setup designs, animal-free human-based modeling frameworks, tissue engineering and drug identification approaches.