Abstract Body: Cardiac fibrosis is a major determinant in diastolic dysfunction, signified by excessive extracellular matrix deposition. This can lead to heart failure, especially heart failure with preserved ejection fraction (HFpEF). Currently, there are no specific treatments targeting HFpEF or its underlying cardiac fibrosis, warranting the urgent need for identifying new and novel therapeutic approaches. Forkhead box protein 1 (Foxe1) is a transcription factor, crucial for cell proliferation and differentiation in thyroid development. Recently we determined that Foxe1 functional loss in the thyroid induced significant fibrosis and increased mast cell number. Interestingly Foxe1 is also abundantly expressed in the heart, yet the effect of Foxe1 functional loss in the heart has never been investigated. We aimed to determine the effect of Foxe1 loss on cardiac structure and function. We hypothesized that the loss of Foxe1 induces cardiac fibrosis by encouraging the transcription of pro-fibrotic genes and inflammatory response. Hearts from 20 week old-tamoxifen induced Foxe1 deficient mice were analyzed for fibrosis and inflammatory cell infiltration. Age matched wild type mice were included as control. Additionally, isolated mouse cardiac fibroblasts were treated with transforming growth factor b1 (TGF-b1, 30ng/mL) as a pro-fibrotic stimulus, and Foxe1 gene level measured. Also, mouse cardiac fibroblasts were cultured for chromatin immunoprecipitation and sequencing (ChIP-Seq) analysis against Foxe1. Our results showed that collagen volume fraction was increased in Foxe1 deficient mice compared to WT, indicative of cardiac fibrosis (1.2±0.35 vs 0.94±0.15%, p=0.038, n=10). Foxe1 deficient mice also had increased mast cell numbers (23.2±3.35 vs 11.6±3.91, p=0.001, n=5) and pro-inflammatory Mac2⁺CD86⁺ macrophages (18.2±4.2 vs 7.6±1.12, p=0.04, n=5) and decreased anti-inflammatory CD206⁺ macrophages (12.4±1.32 vs 103.4±5.99, p<0.0001, n=5) compared to WT. TGF-b1 treatment in mouse cardiac fibroblasts decreased Foxe1 gene level as early as 2 hours (0.66±0.03 vs 1.12±0.05, p=0.01, n=6), indicative of direct and rapid effects of TGF-b1 on Foxe1 gene. ChIP-seq analysis on cardiac fibroblasts revealed that Foxe1 binds to gene related to fibrosis and cell motility such as nhlrc2, clasp2 and mycn. In conclusion, cardiac Foxe1 functional loss induces fibrosis and inflammation in the heart, possibly due to Foxe1 inability to prevent the transcription of some fibrotic genes.