Atrial fibrillation (AF) is the most common cardiac arrhythmia, yet the molecular signature of the vulnerable atrial substrate is not well understood. Here, we delineated a distinct transcriptional signature in right versus left atrial cardiomyocytes (CMs) at baseline, and identified chamber-specific gene expression changes in patients with history of AF in the setting of end-stage heart failure (AF+HF) that are not present in heart failure alone (HF). We observed that human left atrial (LA) CMs exhibit Notch pathway activation and increased ploidy in AF+HF, but not in HF alone. Transient activation of Notch signaling within adult CMs in a murine genetic model is sufficient to increase ploidy in both atrial chambers. Notch activation within LA CMs generated a transcriptomic fingerprint resembling AF, with dysregulation of transcription factor and ion channel genes including Pitx2, Tbx5, Kcnh2, Kcnq1, and Kcnip2. Notch activation also produced distinct cellular electrophysiologic responses in LA versus RA CMs, prolonging the action potential duration (APD) without altering the upstroke velocity in the LA, and reducing the maximal upstroke velocity without altering the APD in the RA. Our results support a shared human/murine model of increased Notch pathway activity predisposing to AF.