Abstract Body: Atrial fibrillation (AF), the most common arrhythmia, is characterized by irregular electrical activity which contributes to electrophysiological and Ca²⁺ cycling remodeling alongside extensive structural modifications, including atrial fibrosis. Fibrosis is marked by excessive extracellular matrix (ECM) deposition impacting AF maintenance and treatment outcomes. While fibrosis is a hallmark of AF in both sexes, sex-specific differences have been observed in ECM protein expression: Young females exhibit lower ECM levels than males, but these levels increase with age in females, likely due to estrogen (E2) loss during menopause. However, the mechanistic basis for E2 protective role against AF fibrogenesis remains unclear. To identify and quantitatively interrogate the mechanisms underlying E2 modulatory role in fibrogenesis during AF, we expanded our previously developed atrial fibroblasts (Fb) model by integrating E2-dependent signaling pathways based on experimental evidence in Fb-related cells. By adjusting E2 initial levels and maximal E2 receptor activation, we developed sex-specific models representing males, premenopausal females, and postmenopausal females. Our findings indicate that ECM levels significantly increase under high profibrotic signals—such as Angiotensin II (AngII) and Transforming Growth Factor β (TGFβ)— in postmenopausal female and male models, but remain unchanged in premenopausal females, aligning with experimental data. Mechanistically, we show that E2 mitigates AngII- and TGFβ-induced ECM upregulation, particularly by the suppression of Plasminogen Activator Inhibitor-1 (PAI1) consistent with experimental data from postmenopausal females. Specifically, E2 counteracts AngII-induced PAI1 by activating Protein Kinase A and inhibiting AngII Receptor Type-1 and Reactive Oxygen Species. Our novel model identifies E2 protective mechanisms against ECM production in Fbs and highlights key regulatory nodes as potential therapeutic targets. Future extensions will incorporate progesterone and testosterone to further refine sex-specific pathways in AF fibrogenesis. Ultimately, this work provides a foundation for developing targeted anti-AF therapies that prevent or reverse atrial fibrosis.