Background: Pulmonary vein isolation (PVI) and antiarrhythmic drug therapy are established treatment strategies to preserve sinus rhythm in atrial fibrillation (AF). However, the efficacy of both interventional and pharmaceutical therapy is still limited. Solid evidence suggests an important role of the cardiac sympathetic nervous system in AF. In this blinded, prospective observational study, we studied left ventricular cardiac sympathetic activity in patients treated with PVI and with antiarrhythmic drugs. Prospectively, Iodine-123-benzyl-guanidine single photon emission computer tomography (123I-mIBG-SPECT) was performed in a total of 23 patients with paroxysmal AF, who underwent PVI (n = 20) or received antiarrhythmic drug therapy only (n = 3), respectively. 123I-mIBG planar and SPECT/CT scans were performed before and 4 to 8 weeks after PVI (or initiation of drug therapy, respectively). For semiquantitative SPECT image analysis, attenuation-corrected early/late images were analyzed. Quantitative SPECT analysis was performed using the AHA 17-segment model of the left ventricle. Results: PVI with point-by-point radiofrequency ablation led to a significantly (p < 0.05) higher visual sympathetic innervation defect score when comparing pre-and post PVI. Newly emerging innervation deficits post PVI were localized predominantly in the inferior lateral wall. These findings were corroborated by semiquantitative SPECT analysis identifying inferolateral segments with a reduced tracer uptake in comparison to SPECT before PVI. Following PVI, patients with an AF relapse showed a different sympathetic innervation pattern compared to patients with sufficient rhythm control. Conclusions: PVI results in novel defects of cardiac sympathetic innervation. Differences in cardiac sympathetic innervation remodelling following PVI suggest an important role of the cardiac autonomous nervous system in the maintenance of sinus rhythm following PVI.