Abstract Body (Do not enter title and authors here): Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with significant morbidity and mortality, yet its molecular mechanisms remain incompletely understood. We identified NFATC1 as a novel AF candidate susceptibility gene, in a multi-generational Utah pedigree with early-onset AF. NFATC1 encodes a calcium-regulated transcription factor essential for cardiac development, but its role in AF pathogenesis is unclear. NKX2-5, a central transcriptional regulator of cardiac conduction and rhythm, has been strongly implicated in AF pathogenesis. We hypothesized that NFATC1 contributes to AF susceptibility by modulating NKX2-5–dependent transcriptional networks and chromatin states in atrial cardiomyocytes. HL-1 cells, a murine atrial cardiomyocyte line, were transfected with Nfatc1-targeting or scramble siRNA(n=3 biological replicates per group), followed by RNA-Seq and ChIP-Seq for Nkx2-5, H3K4me3 (active promoter mark), and H3K27me3 (repressive mark), and Reactome-based pathway enrichment analysis. Publicly available Hi-C data from mouse heart were integrated to evaluate 3D chromatin structure. Nfatc1 knockdown led to widespread transcriptional and epigenomic remodeling. Functional annotation of differentially expressed genes (DEGs) revealed enrichment of pathways involved in cytoskeletal and contractile remodeling, immune and inflammatory responses, and growth factor signaling. Several AF-relevant genes—including Scn5a, Gja1, Kcnq1, and Kcne1—were significantly downregulated, while Nppa was upregulated. ChIP-Seq revealed decreased H3K4me3 enrichment at the promoters of Scn5a, Gja1, Kcnq1, and Kcne1, consistent with transcriptional repression, and reduced Nkx2-5 binding at regulatory regions of each gene. In contrast, Nppa exhibited increased H3K4me3 levels and reduced Nkx2-5 occupancy, suggesting transcriptional activation and disruption of Nkx2-5-regulation. Integration with Hi-C data showed that Scn5a resides within a promoter–enhancer loop connecting its H3K4me3-marked promoter to a distal Nkx2-5–bound region. Reduced Nkx2-5 binding and promoter activation at both loop anchors in Nfatc1-knockdown cells suggest impaired regulation within this pre-existing 3D chromatin domain. These findings reveal that Nfatc1 coordinates atrial gene expression by regulating Nkx2-5 occupancy and chromatin accessibility, and that its loss induces a transcriptional state with molecular features strongly associated with AF.