Mastitis, a prevalent inflammatory disease affecting both humans and animals, imposes significant health burdens globally. Hand.-Mazz. has been traditionally used to treat mammary gland disorders, however, the clinical translation of its crude extracts remains challenging due to the poor bioavailability. Emerging as innovative nanotherapeutic agents, plant-derived extracellular vesicles (PEVs) exhibit enhanced bioavailability, low immunogenicity, and targeted delivery capabilities, making them promising candidates for precision medicine applications. Herein, extracellular vesicles derived from Hand.-Mazz. (TH-EVs) were successfully isolated employing ultracentrifugation and sucrose gradient centrifugation. Subsequently, these physicochemical properties, including particle size distribution and composition analysis, were comprehensively characterized. The anti-inflammatory efficacy and mechanism of TH-EVs were explored in both lipopolysaccharide (LPS)-stimulated murine mammary epithelial cells (HC11) and a murine mastitis model. , TH-EVs reduced TNF-α, IL-6, IL-1β and cellular oxidative stress. , TH-EVs alleviated histopathological damage, decreased myeloperoxidase activity, inhibited T lymphocyte activation, and reduced oxidative stress in mammary tissues. Mechanistically, TH-EVs inhibited the NLRP3 inflammasome, NF-κB and MAPK pathways. This study demonstrates that TH-EVs are a potent natural nanotherapeutic agent for mastitis, with potential for translational applications.