Cell migration plays an important role in physiological and pathological processes in vivo. It has been reported that cells, including skin cells in epidermis and dermis, endothelial cells lining the vessels, and epithelial cells in the mammary glands, are exposed to various mechanical forces such as tensile, compression, shear and electrical stimulation in daily activity. These external stimuli have influence on regulating dynamic cellular responses through mechanotransduction pathways. While extensive studies have been reported on the effects of adhesive proteins and mechanical forces on cellular motility, little is known about how cellular motility is affected by the mechanical properties of their physiological environment. The objective of this study is to investigate the effect of substrate stiffness, ECM protein type, ROCK inhibition and directional force cue on the motility, morphology, spreading area, and shape index of cells. Our results show the stiffness dependent motility behavior with an optimum stiffness where the migration speed is maximized. Different motility behavior of cells on different substrate stiffness most likely arises because cells on soft gels do not adhere strongly enough, reducing the traction forces, while cells on stiff gels adhere too strongly, resulting in decreased migration. At intermediate stiffness, however, cells can attain optimal motility with balanced the cell induced traction force and the reaction force from the environment. However, this sensitive migration to substrate stiffness is suppressed when collagen is used for ECM material. When actin stress fiber formation is suppressed by ROCK inhibitor, average speed of the cells decreases. Through these results, it can be seen that the inhibition of actin stress fiber formation by ROCK inhibitor induces reduction of cell motility, resulting similar tendency of cell motility in all substrate stiffness. However, the biphasic trend of the cell motility is still maintained, ...