Our previous study has shown that three-dimensional (3D) culture decreases mesenchymal stem cell (MSC) size, leading to enhanced trafficking ability and reduced lung vascular obstructions. However, the underlying mechanisms are unclear. In this study, we proposed that 3D culture reduces MSC size by increasing vesicle excretion. Scanning electron microscope showed that 3D culture markedly increased the amount of membrane-bound vesicles on the cell surface. In consistence, tunable resistive pulse sensing quantifying analysis of vesicles in the culture medium indicated that there were higher levels of vesicles in the 3D culture MSC medium. 3D culture significantly lowered the level of actin polymerization (F-actin), suggestive of lowering actin skeleton tension may facilitate vesicle excretion. Indeed, treatment of MSCs with Cytochalasin D or functional blockade of integrin β1 caused increased vesicle secretion and decreased cell sizes. Thus, our results suggest that 3D culture reduces MSC size by increasing vesicle excretion which is likely mediated by lowering cytoskeleton tension. Stem Cells 2018;36:286-292.