Human mesenchymal stem cell (hMSC) derived extracellular vesicles (EVs) have shown therapeutic potential in recent studies. However, the corresponding therapeutic components are largely unknown, and scale‐up production of hMSC EVs is a major challenge for translational applications. In the current study, hMSCs were grown as 3D aggregates under wave motion to promote EV secretion. Results demonstrate that 3D hMSC aggregates promote activation of the endosomal sorting complexes required for transport (ESCRT)‐dependent and ‐independent pathways. mRNA sequencing revealed global transcriptome alterations for 3D hMSC aggregates. Compared to 2D‐hMSC‐EVs, the quantity of 3D‐hMSC‐EVs was enhanced significantly (by 2‐fold), with smaller sizes, higher miR‐21 and miR‐22 expression, and an altered protein cargo (e.g., upregulation of cytokines and anti‐inflammatory factors) uncovered by proteomics analysis, possibly due to altered EV biogenesis. Functionally, 3D‐hMSC‐EVs rejuvenated senescent stem cells and exhibited enhanced immunomodulatory potentials. In summary, this study provides a promising strategy for scalable production of high‐quality EVs from hMSCs with enhanced therapeutic potential.