Abstract: Objective To compare the biological characteristics of three-dimension-spheroid cultured mesenchymal stem cells(3DMSCs) and conventional cultured mesenchymal stem cells (2DMSCs), and explore the potential of 3DMSCs in the cell transplantation in the future. Methods The original MSCs obtained from the abandoned human placenta were cultured in monolayer and 3D spheroid, respectively. Then the morphological characteristics and the dynamic growth were observed under the standard optical microscope. Flow cytometry was used to test the cell apoptosis of the two groups as well as the diameter of cells.Subsequently, the capability of the paracrine effect and anti-inflammation between two groups were tested by RT-PCR and ELISA. Results The 3DMSCs showed a uniform size distribution and inclined to the 3D-spheroid growth. The results of flow cytometry showed that there was no statistically significant difference in the cell apoptosis between these two types of cells (91.6% vs 96%,P=0.234). The diameter size of 3DMSCs were significantly smaller than that of 2DMSCs (10.15 μm vs 16.31 μm, P ＜ 0.001).Most importantly, the RT-PCR showed that the ability to produce the growth factor like VEGF and NT3, and the anti-inflammatory factors including IL-4, IL-10, IL-11, and IL-13 were significantly strengthened in 3DMSCs when compared to the 2DMSCs (all P＜ 0.05).Then we selectively measured the protein level of VEGF, which was also highly expressed in 3DMSCs (222.50±1.23) pg/ml vs(118.07±6.75) pg/ml, P=0.002. Conclusion Compared with 2DMSCs, 3DMSCs are far superior in many aspects of biological characteristics, and should be regarded as the underlying candidate cells for cell transplantation.