Extracellular vesicles (EVs) have drawn attention as promising therapeutic agents whose characteristics resemble their parent cells. However, their practical utility is limited by low EV yields. To address this challenge, cell culture under fluidic flow to enhance EV secretion has been proposed. Yet, the precise mechanism of increased EV production in response to flow conditions has not been thoroughly studied. This study investigated the mechanism of higher release of EVs from mesenchymal stem cells under flow conditions, focusing on the correlation between intracellular calcium ions and EV production. Shear stress was applied to cells through shaking cultures, and stimulated cells showed increased EV production. According to the results, the stimulation of EV secretion is promoted by increasing intracellular calcium ions, primarily due to their transport through the calcium ion channel transport in the plasma membrane, which is induced by shear stress. Furthermore, the study confirmed that the essential characteristics of the EVs released under shear stress remained intact by analyzing individual EVs and assessing their regeneration efficacy of the kidney injury model in vitro. Unveiling the reason for the high production of EVs under shear stress is expected to contribute to the development of EV application research by increasing the reliability of EV utilization.