Potato dry rot, caused by Fusarium species, is a devastating fungal decay that seriously impacts the yield and quality of potato tubers worldwide. Fusarium sulphureum is a major causal agent causing potato tuber dry rot that leads to trichothecene accumulation in Gansu Province of China. Ozone (O3), a strong oxidant, is widely applied to prevent postharvest disease in fruits and vegetables. In this study, F. sulphureum was first treated with 2 mg L-1 ozone for 0, 30 s, 1 min, and 2 min, then inoculated with the potato tubers. The impact of ozone application on dry rot development and diacetoxyscirpenol (DIA) accumulation and the possible mechanisms involved were analyzed. The results showed that ozone treatment significantly inhibited the development of potato tuber dry rot by activating reactive oxygen species (ROS) metabolism and increased the activities of antioxidant enzymes NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) by 24.2%, 13.1%, 45.4%, and 15.8%, respectively, compared with their corresponding control. The activities of key enzymes involved in ascorbate-glutathione cycle (AsA-GSH) of ascorbic peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) also increased by 26.6%, 41.5%, 56%, and 24.1%, respectively, compared with the control group, and their corresponding gene expressions. In addition, ozone treatment markedly suppressed DIA accumulation in potato tubers by downregulating the expression of genes associated with DIA biosynthesis pathway. These results suggest that ozone treatment inhibited the occurrence of potato dry rot and the accumulation of DIA in potato tubers inoculated with F. sulphureum by promoting ROS metabolism and modulating DIA biosynthesis pathway.