Objective To observe the effect of Huangyangning on oxidative stress in dogs with atrial fibrillation. Methods Fifteen beagle dogs were randomly divided into five groups: blank control, model, sham-operated, Huangyangning (0. 24 mg/ kg), and probenecid (7. 58 mg/ kg) groups. A canine atrial fibrillation model was established by placing bipolar electrodes in the right heart ear through femoral vein puncture and connecting a high frequency pacemaker at the caudal end to provide high frequency stimulation. Model establishment was considered successful when atrial fibrillation was confirmed by an electrocardiogram after 1 week of pacing or when an episode of atrial fibrillation was induced during the programmed control. Gavage was started at 1 day after surgery, atrial pacing was started at 1 week after surgery, and samples were collected after 4 weeks. Myocardial fiber changes were observed by Masson staining. Serum levels of angiotensinII (AngII) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were measured by ELISA. Reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured by chemiluminescence. Expression of NADPH oxidase (Nox) subunit genes p22 and p47 was measured by RT-PCR and Western blot. Results Compared with the blank group, serum AngII and NADPH were significantly increased (P< 0. 05) and ROS/ TAC levels were significantly increased (P< 0. 05) in the model group, expression of p22 and p47 genes was significantly increased, and expression of p22 and p47 proteins was increased. There was no significant increase in the serum NADPH level after treatment with Huangyangning or probenecid. ROS/ TAC levels were significantly lower than in the model group (P< 0. 05). Rac-1, p22 and p47 gene expression was significantly lower than in the model group (P< 0. 05). p22 and p47 protein expression was also significantly lower than in the model group (P< 0. 05). Conclusions Huangyangning reduces oxidative stress in dogs with atrial fibrillation, and its mechanism of action may be related to the AngII-NOx-ROS signaling pathway.