Regarded as a strategically crucial material, natural rubber (NR) plays an essential role in our daily life. However, the corresponding rubber products have been facing the problem of aging in which the ozone aging of natural rubber accounts for a crucial position. Based on the concern, the related mechanism of ozone aging for natural rubber was systematically studied in this work. Besides, to deeply understand the mechanism of ozone aging, we selected squalene, a small molecular substance with similar structure to natural rubber, to simulate the ozone aging process and analyze the molecular structure changes of natural rubber. After comprehensive characterization, the obtained results showed that the natural rubber molecular network, during the aging process, was destroyed and three newly-discovered free radicals were generated (•H, •O2−and •C=C), finally resulting in the weakened mechanical properties. We believe this work can provide a more insightful view toward the relevant research on the ozonolysis mechanism and the relationship between structure and performance as well as new ideas for the design of high-efficiency antioxidants for NR.