Background: Pathophysiologic mechanisms underlying lone atrial fibrillation (AF) have not been clearly demonstrated yet. Emerging evidence has indicated that autoimmunity may play a role in the development of AF. Relationship between serum anti-M2-muscarinic receptor autoantibody (anti-M2-R) and anti-β1-adrenergic receptor autoantibody (anti-β1-R) levels and lone paroxysmal atrial fibrillation (PAF) has not been investigated. We aimed to compare anti-M2-R and anti-β1-R levels between lone PAF patients and healthy control subjects. Methods and results: 75 patients with lone PAF (age: 52.80 ± 6.80 years, 53 % male) and 75 healthy control subjects (age: 53.30 ± 6.80 years, 54 % male) were enrolled in the study. Serum anti-M2-R and anti-β1-R levels were measured by ELISA and compared between two groups. Anti-M2-R [142.30 (77.65–400.00) vs. 69.00 (39.48–299.04) ng/mL; p < 0.001) and anti-β1-R [102.56 (65.18–348.41) vs. 44.17 (30.89–158.54) ng/mL; p < 0.001] levels were significantly higher in patients with lone PAF compared to healthy controls. Multivariate regression analysis showed that left atrial diameter (OR: 1.471, p < 0.001), hs-CRP(OR: 1.940, p < 0.001), anti-M2-R (OR: 1.158, p < 0.001) and anti-β1-R (OR: 1.296, p < 0.001) levels were independent predictors for the presence of lone PAF. Using a cut-off level of 101.83 ng/mL, anti-M2-R levels predicted presence of lone PAF with a sensitivity of 94.68 % and specificity of 81.33 %. Anti-β1-R levels predicted presence of lone PAF with a sensitivity of 92.00 % and specificity of 73.30 %, using a cut-off level of 72.16 ng/mL. Conclusion: Our results demonstrated that higher serum anti-M2-R and anti-β1-R levels are associated with lone PAF. Autoantibodies related to autonomic system may play an important role in the development of lone AF.