With the rapid development of stem cell research, the demands for high quality stem cells in cell differentiation studies, cell therapy and clinical applications increase significantly. Human umbilical cord mesenchymal stem cells (hUCMSCs) hold great potentials in these applications due to its wide availability, self-renewal capacity, good pluripotency, and in particular, self-immunomodulation ability. However, the conventional adherent culture system remains challenging in mass production of high-quality stem cells. In this study, we set up a 3D suspension culture system by using spinner flasks added with microcarrier. By optimizing the seeding density and rotation speed, we achieved a cell density as higher as 7×10 ⁸cells/L per vessel. The cultured MSCs had good viability, and the expression levels of the MSC markers were similar to those of 2D-cultured MSCs. After being transferred back into a 2D culture system, the MSCs still maintained normal morphology and proliferation ability. These results indicated that the 3D suspension culture system established in this study provides a fundamental basis for mass production of high-quality MSCs for future research and clinical applications.