BACKGROUND: The therapeutic potential of exosomes derived from mesenchymal stem cells (MSCs) is increasingly recognized in veterinary medicine. This study explored the feasibility of a microcarrier-based three-dimensional (3D) culture system for producing the exosomes (cEXO). Investigations were conducted to enhance production efficiency, ensure stability, and evaluate the therapeutic potential of cEXO for anti-inflammatory applications while assessing their safety profile. RESULTS: The microcarrier-based 3D culture system improved efficient production of cEXO, yielding exosomes with acceptable profiles, including a size of approximately 81.22 nm, negative surface charge, and high particle concentration (1.32 × 10 CONCLUSIONS: This study highlights the potential of a microcarrier-based 3D culture system for scalable cEXO production with robust anti-inflammatory activity, stability, and safety profiles. These findings advance the development of cEXO-based therapies and support their application in veterinary regenerative medicine.