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Although the autonomic nervous system has an evident impact on cardiac electrophysiology and radiofrequency ablation (RFA) is the conventional technique for treating persistent atrial fibrillation, the specific effects of RFA have been insufficiently studied to date. Here, we investigated whether RFA affects neurohumoral transmitter levels and myocardial ¹²³I-metaiodobenzylguanidine (¹²³I-MIBG) uptake. To perform this task, we compared two groups of patients with acquired valvular heart disease: patients who had undergone surgical AF ablation and patients with sinus rhythm. The decrease in norepinephrine (NE) level in the coronary sinus had a direct association with the heart-to-mediastinum ratio (<i>p</i> = 0.02) and a negative correlation with ¹²³I-MIBG uptake defects (<i>p</i> = 0.01). The NE level decreased significantly after the main surgery, both in patients with AF (<i>p</i> = 0.0098) and sinus rhythm (<i>p</i> = 0.0039). Furthermore, the intraoperative difference between the norepinephrine levels in the ascending aorta and coronary sinus (ΔNE) of –400 pg/mL was determined as a cut-off value to evaluate RFA efficacy, as denervation failed in all patients with ΔNE < –400 pg/mL. Hence, ΔNE can be utilized to predict the efficacy of the “MAZE-IV” procedure and to assess the risk of AF recurrence after RFA.