Osteoarthritis (OA) is a degenerative joint disease that affects over 500 million individuals globally, characterized by the degradation of cartilage, subchondral bone sclerosis, and synovitis. A key factor in the progression of OA is synovial inflammation, which is driven by macrophage polarization and inflammasome activation. This inflammation exacerbates cartilage degradation, creating a vicious cycle that accelerates disease progression. Targeting macrophage activity presents a promising therapeutic strategy to alleviate the symptoms and progression of OA.To investigate the role of SEMA6D in macrophage polarization and its potential therapeutic implications for OA, we conducted transcriptomic analysis to explore its regulatory functions. In vitro experiments were performed to assess the effects of SEMA6D overexpression on the expression of ASC and NLRP3, as well as on macrophage (RAW264.7) polarization toward the pro-inflammatory M1 phenotype. In vivo studies were conducted using an OA rat model to evaluate the influence of SEMA6D overexpression on synovial macrophage polarization and the levels of inflammatory mediators, including IL-1β and IL-6.Our transcriptomic analysis indicated that SEMA6D plays a regulatory role in macrophage polarization. Overexpression of SEMA6D resulted in the downregulation of ASC and NLRP3, effectively inhibiting the polarization of macrophages toward the M1 phenotype and downregulate the expression levels of iNOS and IL-6 by more than twofold. In the DMM rat model, SEMA6D overexpression significantly reduced the polarization of synovial macrophages to the M1 phenotype, leading to lower levels of inflammatory mediators such as IL-1β and IL-6, and mitigating cartilage degeneration.SEMA6D exerts a protective effect against OA by attenuating synovial macrophage-mediated inflammatory responses through the inhibition of NLRP3 inflammasome activation and endoplasmic reticulum stress.© 2025 Zhang et al.