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This paper presents the design, fabrication, and the characterization of a stretchable substrate, achieving the change of intracellular calcium ion concentration by mechanical stress. We propose the stretchable substrate integrated with the air chambers of the pneumatic actuator. We have measured the areal strain depending on input pressure and the intracellular calcium ion concentration increase in response to the mechanical stress. The present stretchable substrate is able to provide the areal strain of 5.21% ~ 12.3% for the input pressure of 34.5 kPa ~ 103 kPa. We also verified that the present stretchable substrate is able to stimulate cells by the mechanical stress applied through integrins, showing potential feasible application for monitoring the calcium ion influx caused by the mechanical stress.