To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis (KOA). The rabbits were divided into blank control, model, acupotomy and electroacupuncture (EA) groups, with 12 rabbits in each. Modified Videman's method was used to prepare KOA model. The acupotomy and EA group were given indicated intervention for 3 weeks. The behavior of rabbits in each group was recorded. Subsequently, cartilage-subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography. Compression test was used to detect the mechanical properties of subchondral bone, Western blot and real-time polymerase chain reaction (RT-PCR) were applied to detect the expression of bone morphogenetic protein 2-Smad1 (BMP2-Smad1) pathway in subchondral bone. Compared with the control group, rabbits in the KOA group showed lameness, knee pain, and cartilage degradation; the subchondral bone showed active resorption, the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly. Both acupotomy and EA intervention could increase the thickness of trabecular bone (Tb. Th), the bone volume fraction (BV/TV) and the thickness of subchondral bone plate, reduce the separation of trabecular bone (Tb. Sp), improve the maximum load and elastic modulus of subchondral bone, and effectively delay cartilage degeneration in KOA rabbits. This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway. The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group. Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.