Background— A common single-nucleotide polymorphism (SNP) in the promoter of the Connexin-40 (Cx40) gene GJA5 was suggested to affect Cx40 promoter activity and the risk of atrial fibrillation (AF), but the role of other common Cx40 polymorphisms is unknown. Methods and Results— Eight SNPs within the Cx40 gene region were tested for association with Cx40 levels measured in atrial tissue from 61 individuals. The previously described Cx40 promoter SNP (rs35594137, −44G→A) was not associated with Cx40 mRNA levels. However, a common SNP (rs10465885) located in the TATA box of an alternative Cx40 promoter was strongly associated with Cx40 mRNA expression ( P <0.0001) and displayed strong and consistent allelic expression imbalance in human atrial tissue. A promoter-luciferase assay in cultured murine cardiomyocytes demonstrated reduced activity of the promoter containing the minor allele of this SNP ( P <0.0001). Both rs35594137 and rs10465885 were tested for association with early-onset lone AF (≤60 years of age) in 384 cases and 3010 population control subjects. rs10465885 was associated with the AF phenotype (odds ratio, 1.18; P =0.046). This result was confirmed in a meta-analysis including 2 additional early-onset lone AF case-control cohorts (odds ratio, 1.16, P =0.022). rs35594137 was not associated with the lone AF phenotype in any of the cohorts studied or in a combined analysis. Conclusions— A previously described Cx40 promoter SNP was not found to influence Cx40 expression or risk of AF. We describe an alternate promoter polymorphism that directly affects levels of Cx40 mRNA in vivo and is associated with early-onset lone AF.