Background: Although it is critically important to understand the underlying molecular and electrophysiological changes that predispose to the induction and maintenance of atrial fibrillation (AF), the underlying mechanism of AF is still poorly defined. AF is characterized as the electrophysiological and membrane integrity abnormality of the atrial cells, and claudin-5 (Cldn5), a tight junction protein, may be involved in the pathophysiology of AF, however, the role of Cldn5 in AF is unknown. Methods: Left atrial appendages from the enlarged left atrium were obtained from AF patients undergoing modified radiofrequency ablation maze procedure and normal left atrial appendages were obtained from non-AF donors. Western blot, immunofluorescence, transmission electron microscope (TEM), and proteomics analysis were performed to screen the specific protein expression and signal pathway changes in AF heart tissue vs. non-AF heart tissue. In addition, Cldn5 shRNA or siRNA adeno-associated virus (AAV) were then injected into the mouse left ventricle or added into HL1 cells respectively to knockdown claudin-5 in cardiomyocytes to observe whether the change of Cldn5 influences electrophysiology and affects those protein expressions stem from the proteomic analysis. Mitochondrial density and membrane potential were also measured by Mito tracker staining and JC-1 staining under the confocal microscope in vitro . Results: The protein level of claudin-5 was significantly decreased in cardiomyocytes from the left atrium of AF patients compared to non-AF donors. Proteomics analysis showed that 83 proteins were downregulated and 102 proteins were upregulated in the left atrial appendage of AF patients. Among them, CACNA2D2, CACNB2, MYL2 and MAP6 were dramatically downregulated. KEGG pathway analysis showed these changes would lead to hypertrophic and/or dilated cardiomyopathy. Cldn5 shRNA AAV infection induced-Cldn5 deficiency caused severe cardiac atrophy and arrhythmias in mice. The decreases in both mitochondrial numbers and mitochondrial membrane potential (MMP) were also observed in vitro after Cldn5 knockdown by siRNA. Finally, western blot analysis confirmed the protein level of CACNA2D2, CACNB2, MYL2 and MAP6 were downregulated after Cldn5 knockdown in vivo and in vitro . Conclusions: We demonstrated for the first time the deficiency of Cldn5 in cardiomyocytes in the left atrium of AF patients. The mechanism of AF might be associated with Cldn5 deficiency- associated downregulation of CACNA2D2, CACNB2, MYL2 and MAP6, and mitochondrial dysfunction in cardiomyocytes. Clinical Perspective: What Is New?: This is the first study to find the decreased expression of claudin-5 (Cldn5) with prominent muscle atrophy in the left atrial appendage of atrial fibrillation (AF) patients. Knockdown of Cldn5 in the left ventricle via shRNA adeno-associated virus (AAV) infection caused myocardial atrophy and arrhythmia including ST elevation, replacement of P-waves with f-waves, and absence of P-waves prior to QRS. The protein levels of CACNA2D2, CACNB2, MYL2 and MAP6 were significantly downregulated after Cldn5 deficiency. What Are the Clinical Implications?: The present findings may improve our understanding of the role of Cldn5 in the pathophysiology of AF and provide a new therapeutic target for preventing AF.