Background: Horses and humans are among the few mammals prone to developing spontaneous atrial fibrillation (AF), both suffering from high recurrence rates after treatment. Treatment resistance is often attributed to functional, structural, and metabolic remodeling of the atria. The aim of this explorative study was to evaluate the molecular pathways associated with early stages of AF in horses and to compare the AF proteomes between horses and humans. Methods: We performed data-independent acquisition mass spectrometry on myocardial biopsies collected from the right atrium (RA), left atrium (LA), and left ventricular chamber (LV) in horses with early stages of naturally occurring persistent AF and controls (n=8 in each group). Results: We identified a total of 3400 quantifiable proteins, several of which were differentially regulated between AF horses and controls (268 in RA, 324 in LA, and 284 in LV, p<0.05). Pathway enrichment analyses identified changes in metabolic, contractile, and extracellular matrix (ECM) proteins. Atrial hypocontractility and ECM remodeling were confirmed by echocardiography and histology, respectively. Key proteomic changes in horses with AF overlapped with human AF public datasets, underscoring the translational relevance of the equine AF model. Particularly, contractile proteins, such as cardiac troponins (TNNT2), myosin (MYH6) and tropomyosin (TPM2), along with metabolic regulators as heat shock protein family A (HSPA4) and Proteasome 26S Subunit, Non-ATPase 13 (PSMD13) were shared between horses and humans with AF. Moreover, a significant increase in several ECM glycoproteins was detected in both species, highlighting the importance of early ECM remodeling that extends beyond the fibrotic changes often associated with AF. Conclusion: The horse and human cardiac proteome share similar AF-related changes. Metabolic, functional, and structural protein changes identified in early-stage AF may provide clues to pharmacological targets for preventing AF-associated atrial remodeling and improving treatment success across species.