Background-Collateral damage to the vagal nerve and the upper gastrointestinal (UGI) system during atrial fibrillation ablation has not been systematically evaluated. Methods and Results-We performed a prospective, observational study assessing the effect of atrial fibrillation ablation on the function of the vagus nerve/UGI system. All patients underwent esophageal manometry, gastric emptying study, and sham-feeding test (corresponding to esophageal, gastric, and small intestinal function evaluation, respectively) before ablation (baseline) and subsequently at 24 hours, 90 days, and 180 days after the procedure. In addition, UGI symptom assessment using the patient assessment of upper gastrointestinal disorders-symptom severity index (PAGI-SYM) questionnaire was performed at baseline and during each of the subsequent evaluations. Of the 27 patients enrolled in the study, 9 (33%) patients had abnormal UGI function at baseline; defined as at least one of the 3 abnormal tests. At 24 hours after the radiofrequency catheter ablation, 20 (74%) patients had at least 1 new abnormality on the UGI function tests (P<0.001). New onset esophageal dysmotility, delayed gastric emptying time, and abnormal sham-feeding tests were observed in 13 (48%), 13 (48%), and 9 (33%) patients, respectively. Mean PAGI-SYM scores increased from 7.78±6.6 at baseline to 15.56±13.4 (P=0.002) at 24 hours. New onset abnormalities persisted in 9 (33%) patients at 3 months and normalized in all patients at 6 months. Conclusions-Atrial fibrillation ablation results in functional impairment of the UGI system, including the esophagus, stomach, and small intestine. This impairment is transient and is probably mediated by the injury to the components of the vagal nerve. Clinical Trial Registration-URL: http://clinicaltrials.gov. Unique Identifier: NCT01396356.