Atrial fibrillation is the most common arrhythmogenic disease in humans, with an estimate prevalence of 0.5-5% in the general population, representing a serious burden in terms of morbidity and mortality. Despite its high prevalence, the genetic causes of atrial fibrillation remained largely unknown. Genome-wide association studies have provided novel insights into the genetic bases of AF. Four distinct loci (PITX2, KCNN3, ZFHX3 and IL6R) have been linked to lone AF, and more recently, a GWAS meta-analyses identified another six new loci. Experimental and functional evidence of the involvement of these genes in AF has only been reported for PITX2 describing that Pitx2 haploinsufficiency in mice had an increased susceptibility to atrial arrhythmias and leads to deregulation of Shox2, Tbx3 and Hcn4, as well as sodium and potassium channels. In addition, decreased expression of PITX2 in both right and left atrial appendages of AF patients undergoing surgery was reported. Based on these data, it seems that PITX2 insufficiency leads to cellular, molecular and electrophysiological changes which promote atrial fibrillation. However, it remains to be determined the causal relationship between risk variants and PITX2 expression as well as if PITX2 exert modulatory roles in other genes linked to atrial fibrillation. In this study we surveyed the genetic relationships between distinct AF GWAS associated SNPs in a Spanish AF patient cohort at the allelic, haplotypic and epistatic level. Secondly, we investigated the effect of 4q25 risk variant carriers in the expression of PITX2 as well as other AF GWAS associated genes and thirdly we search for molecular functional links of Pitx2 and those genes contributing to AF pathophysiology in experimental mouse models of Pitx2 insufficiency. Genotype analysis of 130 AF patients demonstrate that only risk variants associated to PITX2, KCNN3, ZFHX3 are significantly linked to AF. Haplotype association was only significant for the 4q25 SNPs. Importantly epistatic relationships were observed for 4q25 (PITX2) and the KCNE1 and KCNN3 risk variants, respectively. Moreover, Pitx2 gain- and loss-of-function analyses demonstrate a pivotal role of Pitx2 regulating Zfhx3, Kcnn3, il6r, Pde4d and Wtn8a expression. Overall these data demonstrate that transcriptional regulation of the homeobox transcription factor Pitx2 is hierarchically controlling multiple aspects leading to cellular, molecular and electrophysiological changes linked to atrial fibrillation.