Abstract Background Post-operative atrial fibrillation (POAF) is associated with worse long-term cardiovascular outcomes. Objectives This study hypothesized that injecting calcium chloride (CaCl2) into the major atrial ganglionated plexi (GPs) during isolated coronary artery bypass grafting (CABG) can reduce the incidence of POAF by calcium-induced autonomic neurotoxicity. Methods This proof-of-concept study randomized 200 patients undergoing isolated, off-pump CABG to CaCl2 (n = 100) or sodium chloride (sham, n = 100) injection. Two milliliters of CaCl2 (5%) or sodium chloride (0.9%) was injected into the 4 major atrial GPs during CABG. All patients received 7-day continuous telemetry and Holter monitoring. The primary outcome was incidence of POAF (≥30 s) in 7 days. Secondary outcomes included length of hospitalization, POAF burden, average ventricular rate during AF, plasma level of inflammatory markers, and actionable antiarrhythmic therapy to treat POAF. Results The POAF incidence was reduced from 36% to 15% (hazard ratio: 0.366; 95% confidence interval: 0.211 to 0.635; p = 0.001). Length of hospitalization did not differ between the 2 groups. POAF burden (first 7 post-operative days), the use of amiodarone or esmolol, and the incidence of atrial couplets and nonsustained atrial tachyarrhythmias were significantly reduced in the CaCl2 group. Heart rate variability data showed a decrease in both high-frequency and low-frequency power in the CaCl2 group with a preserved low-frequency/high-frequency ratio, suggesting that the sympathetic/parasympathetic balance was not perturbed by CaCl2 injection. Conclusions Injection of CaCl2 into the 4 major atrial GPs reduced the POAF hazard by 63%. Inhibition of GP function by Ca-mediated neurotoxicity may underlie the therapeutic effect. (Calcium Autonomic Denervation Prevents Postoperative Atrial Fibrillation; ChiCTR1800019276 )