Introduction and objectives Staphylococcus aureus is an important etiological agent of mastitis, a disease that affects dairy herds causing great economic losses worldwide. Treatment against S. aureus is still ineffective, and its pathogenicity is not yet fully understood. Secretory factors are important components in bacterial pathogenesis, and the secreted nanosized membrane particles, also known as extracellular vesicles (EVs), play a crucial role in intercellular communication. It has been shown that EVs secreted by S. aureus Newbould 305 strain (NWB305), a bovine mastitis isolate, promote cytokine production in vitro, and tissue inflammation and deterioration in vivo, suggesting their role in mastitis pathogenesis. Here, we investigate how NWB305 EVs interact with bovine mammary epithelial cells (bMECs). Materials and methods RNA-seq approach was used to assess bMECs genes modulated by EVs (3 h, 4 h, 8 h and 24 h) and live NWB305 (3 h) in vitro. Transcriptomic results were validated in vivo by RT-qPCR of mice mammary gland tissues. EV uptake by bMECs was evaluated by confocal microscopy and flow cytometry in presence of various transport inhibitors. Results, discussion and conclusion The number of bMECs differentially expressed (DE) genes modulated by EVs increased over time, being 161 modulated at all conditions that included important cytokines and inflammatory genes (eg. NF-κB, IL-6, IL-8, IL-1β). Interestingly, common genes modulated by both EVs and NWB305 after 3 h comprised those with similar and different expression levels, depending on the source of stimulation. EVs also exclusively modulated 125 genes at 3 h, some of which are involved in apoptosis (eg. APOL-3). Finally, approximately half of the genes modulated by EVs at 24 h were also modulated by NWB305 at 3 h, suggesting that EVs stimulation is slower. The modulation of some of these genes by EVs and NWB305 was confirmed in mice mammary glands in vivo. Altogether, our data show that NWB305 EVs may modulate the host immune response with different intensities, exposure times, and by different routes of those of bacteria in different models. Our study uncovers new EV mechanisms of action and potentially functional roles, providing new insights on their impact in staphylococcal pathogenesis.