Abstract Despite extensive research on the cryopreservation of stallion semen, the process still requires improvement. Several diluent additives have been developed to overcome cryopreservation issues, however, semen from valuable individuals often shows poor quality after thawing. Mesenchymal stem cells (MSCs) are regarded as factors that may play an important role in the regenerative process of sperm by secreting extracellular vesicles (EVs) that are actively involved in intercellular communication, tissue repair and cell membrane maintenance. The study aimed to determine the effect of the addition of EV-MSCs obtained from equine adipose tissue on various parameters of cryopreserved stallion semen. All cells met the minimum criteria established by the ISCT, being a characteristic surface profile of the presence of CD90 and lack of MHC II, differentiation into three mesodermal lineages, and adherence. EVs were collected from the expanded cells by ultracentrifugation, and their characteristics were confirmed using nanoparticle tracking analysis (NTA) and flow cytometry to detect CD63 and CD81 antigens. Semen was collected from 11 stallions of the Hucul breed, and each parameter of sperm quality with (25 µg/ml, 50 µg/ml) and without EV addition (motility, morphology and viability) was analyzed. The results showed statistically significant differences in sperm motility, progressive movement and viability following EV treatment. While the amplitude of lateral head displacement (ALH) and morphology parameters did not change, the addition of EVs does not affect these sperm features. The results provide an initial insight into the role of EVs in the maintenance and regeneration of horse sperm after cryopreservation-induced shock.