Objective To design a novel strain loading device for studying the mechanical biology of adherent cells. Methods Based on the technology of substrate deformation loading, the device adopted controllable stepper to cause deformation of the silastic chamber, so as to realize cell loading with multiple units and large strain. The device was developed to test its loading functions. The three-dimensional (3D) models of the silastic chamber were established to simulate the loaded chamber by the finite element technology, and uniformity of the strain field was analyzed. The device applied 5% strain to bone marrow stromal cells (BMSCs) with 0.5 Hz stretch frequency at 2 hours per day for 5 days, and an inverted phase contrast microscope was used to observe the morphology of BMSCs. Results The developed strain loading device for adherent cells in vitro could provide mechanical unidirectional strain up to 50% with three groups of cell loading substrates; within the 10% stain range, the area of uniform strain filed on the silastic chamber remained above 50%, which ensured that the cells were loaded evenly; the morphology of BMSCs was obviously altered, and the direction of arrangement tended to be perpendicular to the loading direction of principal strain. Conclusions The device shows the advantages of reliable operation, wide strain range, adjustable frequency and convenient operation. It can be used to load multiple cell culture substrates at the same time, which provides convenient conditions for the study of cell mechanobiology.