Microvesicles (MVs) released by cells are involved in a multitude of physiological events as important mediators of intercellular communication. MVs derived from mesenchymal stem cells (MSCs) contain various paracrine factors from the cells that primarily contribute to their therapeutic efficacy observed in numerous clinical trials. As nano-sized and bi-lipid layered vesicles retaining therapeutic potency equivalent to that of MSCs, MSC-derived MVs have been in focus as ideal medicinal candidates for regenerative medicine, and are preferred over MSC infusion therapy with their improved safety profiles. However, technical challenges in obtaining sufficient amounts of MVs have limited further progress in studies and clinical application. Of the multiple efforts to reinforce the therapeutic capacity of MSCs, few studies have reportedly examined the scale-up of MSC-derived MV production. In this study, we successfully amplified MV secretion from MSCs compared to the conventional culture method using a simple and efficient 3D-bioprocessing method. The MSC-derived MVs produced in our dynamic 3D-culture contained numerous therapeutic factors such as cytokines and micro-RNAs, and showed their therapeutic potency in in vitro efficacy evaluation. Our results may facilitate diverse applications of MSC-derived MVs from the bench to the bedside, which requires the large-scale production of MVs.