Recent clinical evidence has suggested that interatrial septal (IAS) adiposity contributes to atrial fibrillation (AF). The present study aimed to confirm the usefulness of transesophageal echocardiography (TEE) to estimate IAS adiposity in patients with AF. The histological IAS analysis based on autopsy samples sought to clarify characteristics that underlie the contribution of IAS adiposity to AF. The imaging study analyzed the TEE results in patients with AF (n= 184) in comparison with transthoracic echocardiography (TTE) and computed tomography (CT) results. The autopsy study histologically analyzed IAS in subjects with (n= 5) and without (n= 5) history of AF. In the imaging study, the ratio of interatrial septum adipose tissue (IAS-AT) volume per epicardial adipose tissue (EpAT) volume was greater in patients with persistent AF compared (PerAF) to those with paroxysmal AF (PAF). Multivariable analysis revealed that both TEE-assessed IAS thickness and TTE-assessed left atrial dimension were predicted by CT-assessed IAS-AT volume. In the autopsy study, the histologically-assessed IAS section thickness was greater in the AF group than that in the non-AF group and was positively correlated with the IAS-AT area percentage. In addition, the size of adipocytes in IAS-AT was smaller, compared to EpAT and subcutaneous adipose tissue (SAT). IAS-AT infiltrated into the IAS myocardium, as if adipose tissue split the myocardium (designated as myocardial splitting by IAS-AT). The number of island-like myocardium pieces as a result of myocardial splitting by IAS-AT was greater in the AF group than in the non-AF group and was positively correlated with the IAS-AT area percentage. The present imaging study confirmed the usefulness of TEE to estimate IAS adiposity in patients with AF without radiation exposure. The autopsy study suggested that the myocardial splitting by IAS-AT may contribute to atrial cardiomyopathy leading to AF.