As an essential signaling molecule, nitric oxide (NO) participates in many physiological processes in plant cells, especially the redox system. In order to investigate the role of exogenous NO in alleviating redox damage in peach fruit mitochondria during refrigeration, postharvest peach was treated with NO and c-PTIO (NO scavenger), and the mechanism of NO mitigating mitochondrial injury via antioxidant enzyme and AsA-GSH pathways were explored. The experiment results showed that NO treatment up-regulated transcript levels of related genes involved in the pathways, besides, enzyme activities were increased, which consequently helped strengthen the ability of mitochondria in scavenging reactive oxygen (ROS). Reduced ROS accumulation mitigated mitochondrial damage and maintained mitochondrial morphology and function, maintaining redox balance in peach fruit during cold storage. • NO significantly up-regulates the transcription level of antioxidant enzyme genes. • NO reduces the accumulation of reactive oxygen species in mitochondria. • NO improves the antioxidant capacity of mitochondria.