This study aimed to evaluate the effect of exercise on the superficial zone of the osteoarticular cartilage during osteoarthritis progression. Three-month-old, nine-month-old, and eighteen-month-old Sprague Dawley rats were randomly divided into two groups, moderate exercise and no exercise, for 10 weeks. Histological staining, immunostaining, and nanoindentation measurements were conducted to detect changes in the superficial zone. X-ray and micro-CT were quantitated to detect alterations in the microarchitecture of the tibial subchondral bone. Cells were extracted from the superficial zone of the cartilage under fluid-flow shear stress conditions to further verify changes in vitro. The number of cells and proteoglycan content in the superficial zone increased more in the exercise group than in the control group. Exercise can change the content and distribution of collagen types I and III in the superficial layer. In addition, TGFβ/pSmad2/3 and Prg4 expression levels increased under the intervention of exercise on the superficial zone. Exercise can improve the Young's modulus of the cartilage and reduce the abnormal subchondral bone remodeling which occurs after superficial zone changes. Moderate exercise delays the degeneration of the articular cartilage by its effect on the superficial zone, and the TGFβ/pSmad2/3 signaling pathways and Prg4 play an important role.