Objective This study aimed 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 mechanisms of acupotomy when treating KOA. Methods Forty SD rats were randomly divided into a blank group, a model group, an acupotomy group, and a medication 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 medication group received daily oral celecoxib (10 mg/(kg·d)). Interventions began on day 7 post-modeling, and occurred 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 was used to measure joint space width. Cartilage morphology was evaluated via HE and safranin O-fast green staining. Ligament biomechanical tensile testing was performed. Serum and cartilage tissues were analyzed by ELISA and Western Blot for matrix metalloproteinase-13 (MMP-13) expression. Results (1)Compared with the blank group, the model group showed increased modified Lequesne MG scores, reduced rotarod endurance time, and narrowed joint space (P<0.01). (2)Compared with the model and medication groups, the acupotomy group exhibited lower Lequesne MG scores, prolonged rotarod endurance time (P<0.05), and expanded joint space (P<0.05). (3) The elastic modulus of ligaments in the acupotomy group showed no significant difference from those in the model group but was higher than those in the medication group. Yield strength, maximum strain, and yield-to-tensile strength ratio in the acupotomy group were higher than those in the model and medication groups (P<0.05). (4)HE and Safranin O-Fast green staining revealed minimal inflammatory infiltration in the acupotomy group compared with the model group. Cartilage surfaces in the acupotomy group were smoother than those in the medication group. (5)ELISA showed reduced serum MMP-13 levels in the acupotomy group versus the model group (P<0.01), and no significant differences between levels in the drug and acupotomy groups. (6)Cartilage MMP-13 expression in the acupotomy group was significantly lower than that in the model group (P<0.01) and lower than that in the medication group (P<0.05). Conclusions Acupotomy intervention enhances knee joint stability, improves lower limb mechanical alignment, and suppresses MMP-13 expression in KOA rats.