BACKGROUND: Prior investigations have indicated associations between Knee Osteoarthritis (KOA) and certain inflammatory cytokines, such as the interleukin series and tumor necrosis factor-alpha (TNFα). To further elaborate on these findings, our investigation utilizes Mendelian randomization to explore the causal relationships between KOA and 91 inflammatory cytokines. METHODS: This two-sample Mendelian randomization utilized genetic variations associated with KOA from a large, publicly accessible Genome-Wide Association Study (GWAS), comprising 2,227 cases and 454,121 controls of European descent. The genetic data for inflammatory cytokines were obtained from a GWAS summary involving 14,824 individuals of European ancestry. Causal relationships between exposures and outcomes were primarily investigated using the inverse variance weighted method. To enhance the robustness of the research results, other methods were combined to assist, such as weighted median, weighted model and so on. Multiple sensitivity analysis, including MR-Egger, MR-PRESSO and leave one out, was also carried out. These different analytical methods are used to enhance the validity and reliability of the final results. RESULTS: The results of Mendelian randomization indicated that Adenosine Deaminase (ADA), Fibroblast Growth Factor 5(FGF5), and Hepatocyte growth factor (HFG) proteins are protective factors for KOA (IVW: OR = 0.862, 95% CI: 0.771-0.963,  = 0.008; IVW: OR = 0.850, 95% CI: 0.764-0.946,  = 0.003; IVW: OR = 0.798, 95% CI: 0.642-0.991,  = 0.042), while Tumor necrosis factor (TNFα), Colony-stimulating factor 1(CSF1), and Tumor necrosis factor ligand superfamily member 12(TWEAK) proteins are risk factors for KOA. (IVW: OR = 1.319, 95% CI: 1.067-1.631,  = 0.011; IVW: OR = 1.389, 95% CI: 1.125-1.714,  = 0.002; IVW: OR = 1.206, 95% CI: 1.016-1.431,  = 0.032). CONCLUSION: The six proteins identified in this study demonstrate a close association with the onset of KOA, offering valuable insights for future therapeutic interventions. These findings contribute to the growing understanding of KOA at the microscopic protein level, paving the way for potential targeted therapeutic approaches.