Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.