Background: Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear. Aims: We investigated the link between AF and senescence by comparing the expression of senescence markers (p53 and p16), with prothrombotic and inflammatory proteins in right atrial appendages from patients in AF and sinus rhythm (SR). Methods: The right atrial appendages of 147 patients undergoing open-heart surgery were harvested. Twenty-one non-valvular AF patients, including paroxysmal (PAF) or permanent AF (PmAF), were matched with 21 SR patients according to CHA2DS2-VASc score and treatment. Protein expression was assessed by tissue lysates Western blot analysis. Results: The expression of p53, p16, and tissue factor (TF) was significantly increased in AF compared to SR (0.91 ± 0.31 vs. 0.58 ± 0.31, p = 0.001; 0.76 ± 0.32 vs. 0.35 ± 0.18, p = 0.0001; 0.88 ± 0.32 vs. 0.68 ± 0.29, p = 0.045, respectively). Expression of endothelial NO synthase (eNOS) was lower in AF (0.25 ± 0.15 vs. 0.35 ± 0.12, p = 0.023). There was a stepwise increase of p53, p16, TF, matrix metalloproteinase-9, and an eNOS progressive decrease between SR, PAF, and PmAF. AF was the only predictive factor of p53 and p16 elevation in multivariate analysis. Conclusions: The study brought new evidence indicating that AF progression is strongly related to human atrial senescence burden and points at a link between senescence, thrombogenicity, endothelial dysfunction and atrial remodeling.