Stretchable temperature-responsive cell culture surfaces composed of poly( N-isopropylacrylamide) (PIPAAm) gel-grafted polydimethylsiloxane (PIPAAm-PDMS) were prepared to demonstrate that dual stimulation of temperature and mechanical stress extensively altered graft polymer thickness, surface wettability, and cell detachment behavior. The PIPAAm-PDMS surface was hydrophilic and hydrophobic below and above the lower critical solution temperature, respectively, which was ascribed to the phase transition of PIPAAm chains. When uniaxial stretching was applied, the grafted PIPAAm gel surface was modulated to be more hydrophobic as shown by an increase in the contact angle. Atomic force microscopy observation revealed that uniaxial stretching made the grafted gel layer thinner and deformed the nanoscale aggregates of the grafted PIPAAm gel, implying extension of the PIPAAm chains. The stretched PIPAAm-PDMS became more cell adhesive than the unstretched PIPAAm-PDMS at 37 °C. Furthermore, dual stimulation, shrinking the already stretched PIPAAm-PDMS and decreasing the temperature, induced more rapid cell detachment than only a change in temperature did. Similarly, upon comparison with a single stimulation of a change in temperature or mechanical stress, dual stimulation accelerated cell sheet detachment and harvesting. This new stretchable and temperature-responsive culture surface can easily adjust the surface property to a different cell adhesiveness by appropriately combining each stimulus and enable the fabrication of cell sheets of various species.