<i>Background:</i> Evidence has shown that autoantibodies against M2 muscarinic acetylcholine receptors may play a role in the development of atrial fibrillation. The goal of this study was to evaluate the effects of anti-M2 receptor autoantibodies on rabbit atria in vivo. <i>Methods:</i> Rabbits were immunized monthly with a synthetic peptide corresponding to the M2 receptor. The atrial electrophysiology of the isolated perfused rabbit hearts was studied. Western blots and RT-PCR were performed to determine the expression of the atrial muscarinic receptor and the acetylcholine-activated potassium channel. Atrial tissue was stained with Masson’s trichrome stain for fibrosis detection. <i>Results:</i> Autoantibodies were persistently detected in immunized rabbits. M2 rabbits showed a significantly shorter atrial effective refractory period and a longer intra-atrial activation time than control rabbits. Electrical stimuli induced a significantly larger number of repetitive atrial responses in M2 rabbits. The protein levels of the M2 receptor and GIRK4 were upregulated in M2 rabbits. The mRNA levels of GIRK1 and GIRK4 were also upregulated. Histological examination revealed significantly increased diffuse fibrotic deposition in M2 rabbit atria compared with control rabbits. <i>Conclusion:</i> The M2 receptor autoantibody-positive rabbits showed altered atrial electrophysiology, overexpression of the M2 receptor-I_K,ACh pathway and atrial fibrosis, which indicates that the autoantibodies against M2 receptors may participate in the induction and perpetuation of atrial fibrillation.