Expansion of the cumulus cells (CC) surrounding the oocyte is crucial for growth and maturation of a healthy oocyte, that can be fertilized and develop into an embryo. Factors that are released by CC into the follicular fluid (FF) induce paracrine signaling within the follicular environment, which is essential for proper folliculogenesis. FF is a rich source of proteins, nucleic acids or extracellular vesicles (EVs) which are considered as mediators of intercellular communication within the follicle. EVs influence target cells by interacting with membrane receptors or by internalizing and delivering molecules to cytoplasm or endosomes. Recent studies in cattle have shown an involvement of EVs in controlling cumulus expansion (Hung et al. Biol Reprod. 2015; 93(5):1-9), but this effect has to date not been observed in horses. The aim of this study was, therefore, to evaluate the effect of FF-derived EVs (ffEVs) on equine CC expansion, viability and transcriptome modulations. The material consisted of 43 slaughtered mares’ ovaries, from which 304 cumulus-oocyte complexes (COCs) were obtained. EVs were isolated from small follicles (<20 mm) and characterized as described before (Gabryś et al. Theriogenology 2022; 188:116-124). COCs were matured in vitro (IVM) in the presence (200 µg protein/ml) or absence (control) of ffEVs. For the confirmation of ffEVs internalization, particles were labeled using ExoGlow-protein EV Labeling Kit, added to maturing COCs, which were afterwards imagedusing confocal microscopy. CC expansion was assessed by measuring the COCs diameter before and after IVM and viability test was performed after IVM using staining with ethidium bromide and fluorescein diacetate. Additionally, CC were isolated after 12 hrs of IVM, after which RNA was extracted and cDNA libraries were generated with QuantSeq 3’ mRNA-Seq Library Prep Kit. Transcriptome alterations of CC were investigated with next generation sequencing and bioinformatic analysis. Confocal microscopy images show the internalization of ffEVs by both cumulus cells and oocytes. Supplementation with ffEVs had a significant impact on cumulus expansion in both compacted (Cp, p<0.0001) and expanded (Ex, p<0.05) COCs. There were no differences in diameters of COCs from the control groups measured before and after IVM. The results of viability test indicate a divergent effect of COCs supplementation, as in Cp group ffEVs highly significantly increased (p<0.01), while in Ex decreased (p<0.05) CC viability compared to the control group. Despite the observed differences in CC physiology, transcriptome analysis demonstrate that CC RNA profile is slightly affected in the supplemented group. Nevertheless, the differently expressed genes comprise ones with processes and functions (e.g. cellular and metabolic processes), which may be important for the properties of cumulus and hence the maturation of the oocyte.