Abstract: This study investigated the protective effects of exosome-like nanoparticles derived from Gardenia fruit (GDENs) on PC12 cells damaged by rotenone GDENs were isolated via ultracentrifugation and further purified using sucrose-based density gradient ultracentrifugation. Morphological analysis was examined using transmission electron microscopy (TEM), while their concentration and particle size were determined using nanoparticle tracking analyzer (NTA). Protein concentration was measured by the BCA assay. The protective effects of GDENs were assessed in PC12 cells exposed to rotenone using confocal microscopy with fluorescent dyes, such as 2’,7’-dichlorodihydrofluorescein (DCF) and dihydroethidium (DHE). Results showed that GDENs exhibited a clear membrane structure with a cup-shaped morphology under TEM, with particle sizes ranging from 50 to 150 nm. The RNA content was 9.30±1.06 ng/μL, and the protein concentration was 6.53±0.52 μg/μL. UPLC-MS/MS analysis identified 771 compounds across 12 major categories, predominantly lipids, phenolic acids, and alkaloids. The cell experiments showed that GDENs had good biocompatibility and non-toxicity to PC12 cells. The GDENs could not only be effectively uptaken by PC12 cells, but also reduce the levels of intracellular reactive oxygen species (ROS) and superoxide anion (O₂⁻) damaged by rotenone. This suggested that GDENs had significant effect in reducing oxidative stress. In addition, GDENs could also increase the number of mitochondria in PC12 cells. This elevation might be related to the promotion of mitochondrial biosynthesis and the improvement of its function by GDENs, thereby contributing to the energy metabolism and function maintenance of cells. These findings lay the foundation for the potential applications of GDENs in the field of neuroprotection and antioxidants.