The purpose of this study was to evaluate the effect of low-intensity ultrasound on articular cartilage and subchondral bone alterations in joints under normal and functional disuse conditions during osteoarthritis (OA) progression. Sixty 5-month-old female Sprague-Dawley rats were randomly assigned to six groups (n=10/group): Age-match group, OA group, OA+ultrasound (US) group, Hindlimbs suspension (HLS) group, HLS+OA group, and HLS+OA+US group. The surgical anterior cruciate ligament was used to induce OA in the right knee joints. After two weeks of OA induction, low-intensity ultrasound generated by a 3 MHz transducer with 20% pulse duty cycle and 30 mW/cm2 acoustic intensity was conducted in right knee joints for 20 mins a day, five days a week, and a total of 6 weeks. Then, the right tibias were harvested for Micro-CT, histological and mechanical analysis. Micro-CT results indicated that the thickness and sulfated glycosaminoglycan (sGAG) content of cartilage decreased, but the thickness of the subchondral cortical bone plate and the formation of subchondral trabecular bone increased in the OA group under the normal joint use condition. Furthermore, histological results demonstrated that chondrocyte density and arrangement in cartilage corrupted and the underlying subchondral bone increased during OA progression. These changes were accompanied by reductions of mechanical parameters in OA cartilages. However, less OA symptoms were observed in HLS+OA group under the joint disuse condition. The cartilage degeneration and subchondral bone sclerosis were alleviated in the US treatment group, especially under normal joint use condition. In conclusion, low-intensity ultrasound could improve cartilage degeneration and subchondral sclerosis during OA progression. Also, it could provide a promising strategy for future clinical treatment for OA patients.