Mesenchymal stem cells (MSC), nonhematopoietic progenitor cells are capable of differentiating into multiple lineage of the mesenchyme such as bone, cartilage, tendon, fat, heart, muscle, and brain, in vitro and in vivo. Although autologous MSC have some advantageous factors, MSC have very poor replicative capacity. Previous studies indicated that growth factors but also cell seeding density made an effect on expansion of MSCs. In this study, we examined the growth rate and adhesion of MSC in tissue culture coated with a basement membrane-like extracellular matrix. When self-renewal of MSC were compared between 2-D and 3-D, the growth rate of MSCs on 3-D matrix was highest among plastic, Fibronectin- and 3-D matrix plate but also attachment of MSCs on 3-D matrix was 6-fold greater than that on plastic. MSCs plated into 3-D matrix showed colocalization of α5 integrin and αV integrin. This study may reflect that the extracellular matrix had greater effect on the expansion of MSCs because this distinctive in 3-D matrix adhesion differ in localization and function from classically described in vitro adhesion.