Microenvironment of the extracellular matrix can influence cellular responses through alternation of initial attachment and induce production of new tissue. To study the effect of such microenvironment on the relationship of cell cytoskeletal shape and its biological behaviors such as adhesion, proliferation and differentiation, we designed a patterned strip line of fibronectin on self assembled monolayers via microcontact printing. The physiological behavior of human mesenchymal stem cell (hMSC) on defined micro-patterns of fibronectin was evaluated after 4 h and 2 days of culture. Initial adhesion of hMSCs on a substrate with pattern spacing of 11 μm was stabilized faster than that on other substrates. Ratio of proliferating hMSC on 5 and 11 μm substrate constantly maintained a high rate. hMSCs on 5 and 11 μm substrate could adhere to substrate as spreading from fibronectin pattern line to several and lateral fibronectin pattern line. Their nucleus area could represent artificial increase by widely spreading on several fibronectin pattern lines. On the contrary to this, ratio of proliferating hMSC on 20 μm substrate constantly maintained a low rate less than even control and 0 μm substrate without fibronectin pattern. Tiny nucleus caused narrow and elongated hMSC morphology on 20 μm substrate gave the negative effect on the cell adhesion and proliferation. However, hMSCs on 20 μm substrate possessed not only slightly increased value of GO/G1 phase but also down regulation of CD marker expression compared with other groups. These results show initial adhesion and morphology of hMSC could regulate specific cellular behavior of hMSC.