Abstract Body (Do not enter title and authors here): Background: Tumor endothelial marker 1 (TEM1 or CD248) is a transmembrane protein found in activated mesenchymal cells during embryo development. Its expression level becomes very low or undetectable in adult tissue. Re-expression of TEM1 in fibroblasts has been reported in hepatic, renal, and pulmonary fibrosis. We evaluated atrial TEM1 expression in atrial fibrillation (AF) and its relation with atrial fibrosis. Methods and Results: All data were presented as mean±standard error. Left atrial (LA) tissues were collected from 30 patients (pts) (mean age 64.0±1.6 yrs, male 76.7%) with AF underwent cardiac surgery (mitral and/or tricuspid valve surgery 96.7%) and maze procedure with LA appendage excision. Immunofluorescence (IF) staining showed TEM1 expressed in atrial cardiac fibroblasts (CFs) in the LA tissue of AF pts, but not in normal LA tissue (US Biomax). Primary human CFs were directly isolated from the LA tissue. IF staining showed TEM1 expression in the isolated CFs from AF pts but not in normal human CFs (ScienCell). Western blot could detect TEM1 expression in the LA tissues of all 30 AF pts. Sirius Red staining was used to visualize collagen and atrial fibrosis was quantified using a pixel-based method (Adobe Photoshop) by calculating the total numbers of red pixels and the percentage (%) of red pixels to total pixels. Atrial fibrosis was increased in AF pts compared with normal control (total number of red pixels: 160740±13066 vs. 50863±6357, % of red pixels to total pixels: 11.4±0.8 vs. 3.8±0.2%, all p<0.001). There was a positive correlation between the expression level of TEM1 (ratio of TEM1/actin band density in western blot) with the severity of atrial fibrosis, including the total numbers of red pixels (r = 0.47, p = 0.01) and the % of red pixels to total pixels (r = 0.48, p = 0.01). TEM1 and galectin expression levels had positive correlation (r = 0.60, p < 0.01). There were no correlations of atrial TEM1 expression with the LA diameter and CHA2DS2-VASc score. In vitro study showed TEM1 knockdown in cultured CFs with siRNA reduced cell proliferation (scrambled- vs TEM1-siRNA, BrdU absorbance: 1.84±0.05 vs 1.50±0.07, p<0.05), increased starvation-induced cell apoptosis (cell death ELISA absorbance: 0.69±0.11 vs 2.88±0.05, p<0.05) and decreased cell migration. Conclusions: There is increased TEM1 expression in atrial CFs in AF and its expression level is associated with atrial fibrosis. TEM1 expression changes the cell behaviors of CFs.