BACKGROUND: Tuina therapy has demonstrated its potential in modulating autophagy-related factors in knee osteoarthritis (KOA); however, its core therapeutic targets and specific mechanisms require systematic elucidation through interdisciplinary research. OBJECTIVE: This study investigated the mechanism by which Tuina alleviates KOA progression using multidimensional approaches, including Mendelian randomization (MR), in vivo experiments, and machine learning. METHODS: Genetic data from genome-wide association studies of 60 cytokines and KOA were analyzed using MR analysis to identify autophagy-related factors significantly associated with KOA. A KOA rat model was established via intra-articular injections of L-cysteine-activated papain solution into the right knee. The key autophagy-related cytokines identified by MR were validated using enzyme-linked immunosorbent assay (ELISA). Cartilage degeneration and autophagic activity were assessed through histological evaluation via safranin O/fast green (SO/FG) staining and ultrastructural analysis via transmission electron microscopy (TEM). A Support Vector Machine (SVM) algorithm was used to conduct a secondary analysis of the experimental dataset to predict comprehensive therapeutic effects and synergistic correlations among the indicators. RESULTS: MR analysis revealed causal relationships between eight cytokines and KOA at genetically determined levels, with seven exhibiting autophagy-related regulatory properties. Tuina significantly alleviated pain and improved motor function. SO/FG staining and TEM revealed reduced cartilage destruction, increased cartilage thickness, and decreased chondrocyte autophagy. ELISA revealed that Tuina downregulated interferon gamma, parathyroid hormone, interleukin (IL)-1β, matrix metalloproteinase-3, and IL-17 levels in the cartilage while upregulating osteocalcin and transforming growth factor-β1 levels. Furthermore, Tuina demonstrated superior comprehensive and classified curative effects compared with celecoxib. IFN-γ and PTH were strongly correlated with both relative autophagy level and cartilage area ratio. Autophagy-related cytokines had the strongest correlation with the degree of cartilage degeneration. CONCLUSION: Tuina therapy alleviates pain and functional impairment in KOA by suppressing chondrocyte autophagy and delaying cartilage degeneration. This mechanism may involve the regulation of autophagy-related cytokines.