Cells cultured in vitro using traditional substrates often change their behavior due to the lack of mechanical deformation they would naturally experience in vivo. To mimic the in vivo mechanical environment, we design temperature-responsive hydrogels with patterned surface creases as dynamic cell stretching devices. A one-step photolithographic method is first employed to pattern integrin-binding peptides on the gel, causing single cells or several-cell clusters to adhere to the surface in registry with creases. A variety of crease patterns are prescribed on a single substrate, enabling the mechanical deformation of cultured myoblast cells with different strain states and achieving tensile strain as high as 0.2. As creases provide large amplitude local deformation of the gel surface without the need for macroscopic deformation, can be formed on gels covering a wide range of modulus, and can be actuated using a variety of stimuli, they hold the potential to enable the design of high throughput and versatile platforms for mechano-biological studies.