Osteoarthritis (OA) is a degenerative osteoarticular pathology that can affect all the synovial joints, and is marked by the gradual deterioration of hyaline cartilage. This disorder compromises the well-being of horses, and leads to early career retirement, resulting in significant financial burdens for the equine industry. Over the last few years, the use of the immunomodulatory and cartilage regenerative potentials of mesenchymal stromal cells (MSCs) have emerged, but has also raised several concerns. As MSCs therapeutic properties are mainly driven by their secretome, exosomes derived from MSCs appear to be a promising therapeutic strategy for OA management. However, to our knowledge, the purification and characterization of equine exosomes were not well documented and needed more in-depth investigation. Here, we first purified exosomes from MSCs by membrane affinity capture (MAC) and size-exclusion chromatography (SEC), and validated a panel of equine exosomes markers (CD9, CD63, CD81, CD82, Alix and Tsg101) for their characterization. In addition, we showed that purified exosomes were intact and could be internalized by equine articular chondrocytes (eACs). Through the comparison of the two isolation methods, exosomes from MAC exhibited superior results in enhancing the neo-synthesis of a hyaline-like matrix by eACs. This enhancement was highlighted by the modulation of collagen levels, an increase in Pcna expression, and a decrease in Htra1 synthesis. However, due to unexpected effects induced by the MAC elution buffer on eACs, an additional ultrafiltration step was incorporated into the isolation protocol. Furthermore, our investigation revealed that exosomes derived from MSCs primed with equine pro-inflammatory cytokines, such as IL-1β, TNF-α, or IFN-γ, improved the hyaline-like phenotype of eACs, particularly IL-1β and TNF-α. Collectively, these findings underscore the significance of the exosome purification method and the potential benefits of pro-inflammatory cytokine priming to enhance the therapeutic efficacy of exosomes for the treatment of equine OA.