Background: Atrial septal defect (ASD) is the third common cause of congenital heart disease. Amplatzer septal occluder (ASO) is a double-disc, self-expandable, occlusion device, which is made of nickel-titanium-alloy and approved for percutaneous closure of secundum ASD. However, the efficacy of ASO on ASD in animals remains unclear. This study intends to investigate the effect and molecular mechanism of ASO treatment on ASD closure in beagles. Methods: mRNA and protein expression of HERG, Mink, Kir2.1 or Kir2.3 genes in the right atrium tissues of beagles was analyzed by quantitative real-time PCR or Western blot. The localization of Connexin 40 (CX40), CX43 or Collagen type I (Col I) in the right atrium tissues was determined by immunohistochemistry assay. Fibrosis of atrial tissues was examined by Masson trichrome staining. Electrophysiological parameters of atrial tissues including ERP HRA, CSp or CSd were profiled using a programmable electrical stimulator. Results: ASO treatment significantly enhanced both mRNA and protein expressions of HERG, Mink, Kir2.1 and Kir2.3 in the right atrium of beagles in comparison with ASD without ASO treatment. ASO markedly suppressed CX40, CX43 or Col I expression in the tissues. Measurement of electrophysiological parameters demonstrated that ASO treatment significantly reduced ERP HRA, CSp or CSd in treated beagles in comparison with the ones in ASD group. Moreover, ASO treatment significantly inhibited atrial wall fibrosis in treated beagles compared to the ones in ASD group. Conclusions: ASO has potent therapeutic effect to treat ASD in beagles.