Atrial fibrillation (AF) is the most common cardiac arrhythmia. The genetics of AF are complex, but recent GWAS have revealed a number of AF risk loci, including TBX5 . TBX5 plays a critical role in heart development, and continues to be expressed in the adult atria. We assessed the role of Tbx5 in the adult heart by removing Tbx5 from the mouse at 6 weeks-of-age. Adult Tbx5 mutant mice developed spontaneous sustained atrial fibrillation, characterized by surface electrocardiogram (ECG), intracardiac ECGs, and ex vivo whole heart optical mapping. Tbx5 mutant isolated cardiomycytes showed prolonged action potentials, disrupted calcium handling, and abnormal automaticity. Tbx5 mutant atrial gene expression analysis revealed down-regulation of numerous conduction system effectors genes including Scn5a, Ryr2, Gja1, Atp2a2 , Dsp , and numerous potassium channels. We also observed Tbx5 dependence of the transcription factor gene Pitx2 , the number one locus associated with AF by GWAS. We identified an enhancer upstream of PITX2 that interacts with the PITX2 promoter by 4C and whose activity is TBX5 dependent and modulated by common SNP rs1906595, which alters a canonical TBX5 binding site. We also identified cis-regulatory elements at Scn5a, Ryr2, Gja1, Atp2a2 , and Dsp , locus co-regulated by Tbx5 and Pitx2. We describe a TBX5-driven atrial molecular network including Pitx2 that determines atrial conduction properties.