BACKGROUND: While studies aimed at overcoming ischemia-reperfusion (IR) injury using various materials are becoming popular, studies using botulinum toxin type A (BoNTA) are still limited. This study tested the hypotheses that BoNTA can protect flaps from IR injury by inhibiting the NADPH oxidase system and suppressing ROS (reactive oxygen species) production. MATERIAL AND METHODS: The subjects were Sprague-Dawley rats (n = 76). In 4 rats, the effects of different dose of BoNTA on superoxide production was evaluated through lucigenin enhanced chemiluminescence assay (LECL) using SD rats' thoracic aorta ring. In 60 SD rats, The BoNTA and normal saline-pretreated superficial inferior epigastric artery (SIEA) flaps were clamped for 0, 1, and 4 hours, and reperfused. On the 5th day after the opeartion, well-maintained flaps were grossly inspected, survival rates were analyzed, and histological analysis was also performed. In 12 rats, after making IR injury through the same model, SIEA flap segments and femoral vessels were obtained, and ROS production was evaluated through LECL and dihydroethidium (DHE) staining. RESULTS: In LECL, the experimental group produced a smaller amount of superoxide than the control group through NADPH oxidase inhibition (p < .05). There was no significant difference between the experimental and control group in the 0, and 1 hour IR groups, but the experimental group (90%) showed a higher survival rate than the control group (60%) in the 4 hours IR group (p = .028). In the measurement of ROS production through LECL and DHE staining, there was no significant difference in the 0, and 1 hour IR groups, but a significant difference was shown in the 4 hours IR group in both the SIEA flaps and femoral vessels (p < .05). SUMMARY: This study verified hypothesis that BoNTA can protect flaps from IR injury by inhibiting the NADPH oxidase system and suppressing ROS production. Based on this research model, future research should be expanded into studies on subtypes or subunits of NADPH oxidase, and the findings from the present study are expected to contribute and lead to clinical studies on BoNTA, which has already been proven to be clinically safe.