Aims: The purpose of this study was to evaluate the contribution in the acute vagal denervation after ablation with the second generation cryoballoon (CB-A) in each distinct pulmonary vein (PV) by the means of external cardiac vagal stimulation (ECVS), by positioning a catheter in the internal jugular vein, in a cohort of 60. Methods: Sixty patients, 50 starting from the left superior pulmonary vein (LSPV) and 10 from the right superior pulmonary vein (RIPV) with symptomatic paroxysmal atrial fibrillation (PAF), having undergone ECVS before the first and after each PV ablation by means of CB-A ablation were included. Results: The ECVS performed pre-ablation provoked cardioinhibitory responses in all cases with mean pause duration of 10251.83 ms +/- 2826.23 ms. At the end of the procedure, the vagal reactions (VR) were significantly diminished. Specifically, compared against the initial pause, responses were 8957.06 +/- 2711.66 ms (p < 0.01) after left superior PV, 10017.36 +/- 9127.0 ms (p = 0.88) after left inferior PV, 6020.16 +/- 3780.709 ms (p < 0.001) after right inferior PV and 1687.5 +/- 2183.7 ms (p < 0.001) after right superior PV. Noteworthy, if starting with ablation in the RSPV, VR was immediately reduced by 90.34%, 990.7 +/- 379.78 ms (p < 0.001) if compared to baseline response. Conclusion: Although not directly targeting the ganglion plexuses, AF ablation with the CB-A causes a significant acute parasympathetic denervation. The RSPV showed to be associated to the most significant reduction of acute parasympathetic denervation.