Patterning of surface using soft lithography is useful tools for regulating cellular morphology as well as function. Microcontact printing technology was used for patterning extracellular matrix (ECM) protein in this study. This pattern was used for controlling cellular adhesion as well as morphology of human mesenchymal stem cell. Polydimethylsiloxane (PDMS) molds was designed for controlling cellular adhesion by varying stripe width from 5 to 50 μm and fixing spacer to 20 μm. Glass surface was coated with extracellular matrix proteins such as fibronectin and vitronectin with different PDMS molds. Human mesenchymal stem cells were cultured on the patterned surfaces. Cells were aligned on the surfaces by changing cellular morphology. Actin filaments were aligned according to the protein stripe on the surfaces. The length of the actin fiber is longer as the stripe width is thinner. Proteins for focal contact such as vinculin, paxillin, and FAK were localized only on the ECM patterned surfaces. Especially, paxillin and FAK playing key role for focal adhesion were colocalized on the patterned surfaces. This study provides the controlling localization of focal adhesion proteins as well as morphology of human MSCs on the patterned surfaces. For further study, the effects of the localization of focal contacts proteins of MSCs to the stemness and differentiation will be investigated.