A novel method for the fabrication and the use of simple uniform poly(dimethylsiloxane) PDMS substrates for controlling cell motility by a mechanical gradient is reported. The substrate is fabricated in PDMS using soft lithography and consists of a soft membrane suspended on top of a patterned PDMS substrate. The difference in the gradient stiffness is related to the underlying pattern. It is shown experimentally that these uniform substrates can modulate the response of cell motility, thus enabling patterning on the surfaces with precise cell motility. Because of the uniformity of the substrate, cells can spread equally and a directional movement to stiffer regions is clearly observed. Varying the geometry underlying the membrane, cell patterning and movement can be quantitatively characterized. This procedure is capable of controlling cell motility with high fidelity over large substrate areas. The most significant advance embodied in this method is that it offers the use of mechanical features to control cell adhesion and not topographical or chemical variations, which has not been reported so far. This modulation of the response of cell motility will be useful for the design and fabrication of advanced planar and 3D biological assemblies suitable for applications in the field of biotechnology and for tissue-engineering purposes.