Subchondral bone (SB) is recognized as a key factor in normal joint protection, not only does it provide a shock absorbing and supportive function for the cartilage, but it may also be important for cartilage metabolism. Mechanical loading is considered to be a critical regulator of skeletal homeostasis, including bone and cartilage. It is suggested that both cartilage and bone may respond to mechanical loading in an intensity-dependent manner. In this report, we have discovered that the subchondral plate became thicker with higher bone mineral density (BMD) and lower porosity, while trabecular bone became more plate-like and denser with higher BMD in high-intensity running (HIR) group. Further, HIR led to highly remodeled, less mineralized, and stiffer subchondral plate and trabecular bone. On the contrary, low-intensity running and moderate-intensity running failed to result in considerable changes in microstructure, composition and hardness. Our findings suggested that running affects SB in an intensity-dependent manner. In addition, HIR may induce change in organization and composition of SB, and consequently alter its mechanical properties. HIR-induced "brittle and stiff" SB may adversely affect the overlying articular cartilage.