AbstractAtrial fibrosis is involved in the progression of atrial fibrillation (AF). miR-499-5p is the most downregulated microRNA in arrhythmogenic cardiomyopathy hearts. Sry-related high-mobility-group box 6 (SOX6) is associated with apoptosis, inflammatory responses, and fibrosis. This study investigated the mechanism of miR-499-5p in ameliorating AF rats by regulating SOX6. AF rat models were established by injecting Ach–CaCl2mixture, and the rats were treated with Lv-miR-499-5p/oe-SOX6/si-SOX6 before modeling. AF duration was recorded using electrocardiogram. miR-499-5p and SOX6 expression levels in the myocardium were determined by reverse transcription-quantitative polymerase chain reaction. The binding of miR-499-5p and SOX6 was validated. The atrial fibrosis degree and cardiomyocyte apoptosis were assessed using the Masson and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining methods. Levels of SOX6, atrial fibrosis markers (collage I/α-SMA/TGFβ1), cell cycle-related proteins (p21/CDC25/Cyclin B1), and cell senescence markers (SA-β-gal/γ-H2AX) were measured using Western blotting and immunohistochemistry. miR-499-5p was downregulated and SOX6 was upregulated in AF rats. miR-499-5p overexpression shortened the AF duration, alleviated atrial fibrosis, and decreased collage I/α-SMA/TGFβ1. miR-499-5p targeted SOX6 to ameliorate atrial fibrosis. AF rats exhibited increased p21/CDC25/Cyclin B1/SA-β-gal/γ-H2AX levels and raised cardiomyocyte apoptosis. SOX6 silencing downregulated p21 and alleviated cardiomyocyte cycle arrest, cell senescence, and apoptosis in AF rats. Shortly, miR-499-5p suppresses atrial fibrosis and cardiomyocyte senescence by targeting SOX6 and downregulating p21, thus mitigating AF in rats.