Objective To observe the effects of Yin-Yang balancing acupotomy intervention on knee joint function and lower limb biomechanics in a rat model of knee osteoarthritis (KOA), and to explore the mechanism of acupotomy in treating KOA. Methods Forty SD rats were randomly divided into a blank group, model group, acupotomy group, and drug group. Except for the blank group, KOA models were established by injecting a mixed solution of 4% papain and 0.03 mol/L L-cysteine into the left knee joint cavity. The acupotomy group received Yin-Yang balancing acupotomy interventions targeting the medial/lateral collateral ligaments and patellar ligament. The drug group received daily oral celecoxib (10 mg/kg·d). Interventions began on day 7 post-modeling, once weekly for 4 weeks. All rats were assessed pre- and post-intervention using the modified Lequesne MG knee joint grading system and rotarod fatigue test. Post-intervention, in vivo DR imaging measured joint space width. Cartilage morphology was evaluated via H&E and Safranin O-Fast Green staining. Ligament biomechanical tensile testing was performed. Serum and cartilage tissues were analyzed by ELISA and Western blot (WB) for matrix metalloproteinase-13 (MMP-13) expression. Results ①Compared to the blank group, the model group showed increased modified Lequesne MG scores, reduced rotarod endurance time, and narrowed joint space (P < 0.01).②Compared to the model and drug groups, the acupotomy group exhibited lower Lequesne MG scores, prolonged rotarod endurance time (P < 0.05), and expanded joint space (P < 0.05).③The elastic modulus of ligaments in the acupotomy group showed no significant difference from the model group but was higher than the drug group. Yield strength, maximum strain, and yield-to-tensile strength ratio in the acupotomy group were higher than in the model and drug groups (P < 0.05).④H&E and Safranin O-Fast Green staining revealed minimal inflammatory infiltration in the acupotomy group compared to the model group. Cartilage surfaces in the acupotomy group were smoother than in the drug group.⑤ELISA showed reduced serum MMP-13 levels in the acupotomy group versus the model group (P < 0.01), with no significant difference between the drug and acupotomy groups.⑥Cartilage MMP-13 expression in the acupotomy group was significantly lower than in the model group (P < 0.01) and lower than in the drug group (P < 0.05).Conclusion Yin-Yang balancing acupotomy improves knee joint stability, enhances lower limb biomechanics, and reduces MMP-13 expression in KOA model rats.