Cardiomyocyte senescence drives cardiovascular disease, underscoring the need to define its molecular mechanisms. The role of cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in this process remains unclear, particularly regarding its expression and function. Atrial tissues were collected from patients with sinus rhythm or atrial fibrillation (AF) of varying durations. CFTR was downregulated in AF patients and negatively correlated with p16, p21, and p53. Myocardial aging models were established using D-galactose (D-gal) in both mice and neonatal mouse cardiomyocytes (CMs). In both animal and cellular models, D-gal increased SA-β-gal positivity and senescence markers while decreasing CFTR. Overexpressing CFTR reduced D-gal-induced elevations in p16, p21, p53, and malondialdehyde (MDA), and restored superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities. Mechanistically, CFTR alleviates mitochondrial oxidative stress damage by enhancing plasma membrane Ca2+ ATPase (PMCA) activity to reduce cytoplasmic Ca2+ levels. Furthermore, we identified USP45 as a direct binding partner of CFTR, which deubiquitinates CFTR by specifically targeting K48-linked chains and the K688 residue. CFTR knockdown exacerbated D-gal-induced senescence and mitochondrial oxidative stress, which was rescued by USP45 overexpression. In conclusion, this study reveals a novel mechanism in which USP45-mediated deubiquitination of CFTR mitigates cardiomyocyte senescence and mitochondrial oxidative stress, offering a targeted intervention against age-related cardiovascular diseases.