Despite intervertebral cartilage endplate exerts a pivotal role in disc degeneration, little is known about changes in stem cells derived from cartilage endplate (CESCs) and their regenerative potential with disc degeneration. Biomechanical extracellular matrix cues have recently been shown to play a crucial role on stem cell behavior, but with the process of disc degeneration, the effect of matrix stiffness on CESCs behavior is unknown. This work aims at understanding the CESCs behavior under mechanical cues with the process of disc degeneration. Atomic force microscopy (AFM) detects CEP matrix stiffness with different degeneration degree. In order to better understand the contribution of matrix stiffness to CESCs proliferation and differentiation, a polyacrylamide gels was developed with different Young’s elastic modulus (EY) that mimicked the native stiffness of CEP tissue with different degeneration degree. Culturing CESCs on gels that mimicked the stiffness of young CEP tissue (normal, 35.2kPa) expressed highest genes responsible for stem cells than other gels and became quiescent. But as the gels that mimicked the mild degeneration of CEP tissue (72.5kPa), the cells significantly unregulated chondrogenic-specific genes. When the gels that mimicked the severe degeneration of CEP tissue (102.7kPa), the cells upregulated osteogenic-specific genes. As substrate stiffness increased, the cells became more spread and increased proliferation capacity. Together these data imply that with the process of disc degeneration, mechanical cues keep stem cells pools quiescent at early stage, promote repairing and restrain degeneration at middle stage (mild degeneration degree), accelerate degeneration at late stage (severe degeneration degree).