OBJECTIVE: To observe the effect of Gubi Powder, an in-hospital prescription, on inflammatory factors and Notch3/Hes1 and Wnt/β-catenin signaling pathways in rabbits with knee osteoarthritis model, providing new drug ideas for clinical treatment. METHODS: Forty New Zealand rabbits were divided into sham operation group, model group, positive group (diclofenac potassium gel) and Gubi Powder group, with 10 rabbits in each group. The modified Hulth method was used to construct the rabbit knee osteoarthritis model, which lasted for 8 weeks. After modeling, normal saline was applied to the knee joints of the sham operation group and model group, diclofenac potassium gel was applied to the positive group, and Gubi Powder was applied to the Gubi Powder group, 8 hours a day, for 1 week. After administration, animal activity and behaviors were evaluated and scored; serum levels of prostaglandin E2, cyclooxygenase-2, interleukin 1β and tumor necrosis factor α were determined by ELISA; pathological changes of cartilage tissue were observed by hematoxylin-eosin staining and Safranin-O staining; protein levels of matrix metalloproteinases 1, 2, 3, and 13 in cartilage tissue of the knee joint were determined by immunohistochemistry; and protein levels of Notch3, Hes1, Wnt5a, β-catenin, Bcl-2, Bax, and Caspase-3 in cartilage tissue of the knee joint were determined by western blot assay. RESULTS AND CONCLUSION: Compared with the sham operation group, animals in the model group had difficulties in walking, serious cartilage injury in the knee joint, significantly elevated levels of serum prostaglandin E2, cyclooxygenase 2, interleukin 1β and tumor necrosis factor α (P < 0.05), significantly increased protein expression levels of matrix metalloproteinases 1, 2, 3, and 13, Bax, Caspase-3, Wnt5a and β-catenin in cartilage tissue (P < 0.05), and decreased protein expression levels of Notch3, Hes1, and Bcl-2 (P < 0.05). Compared with the model group, the walking condition of animals in the positive group and Gubi Powder group was improved, the serum levels of prostaglandin E2, cyclooxygenase-2, interleukin 1β and tumor necrosis factor α were decreased, cartilage damage of the knee was alleviated, the protein expression levels of matrix metalloproteinases 1, 2, 3, and 13, Bax, Caspase-3, Wnt5a and β-catenin in cartilage tissue were reduced (P < 0.05), and the protein expression levels of Notch3, Hes1, and Bcl-2 were increased (P < 0.05). To conclude, Gubi Powder can alleviate the symptoms of knee osteoarthritis in rabbits, improve the pathological changes of the cartilage, and reduce inflammatory factor levels in serum. The mechanism of action may be related to the inhibition of Notch3/Hes1 and Wnt/β-catenin signaling pathways. [ABSTRACT FROM AUTHOR]