Objective <p>Although synovial inflammation is recognized as a factor in the progression of osteoarthritis (OA), the specific role of fibroblast-like synoviocytes (FLS) in cartilage degradation has yet to be fully elucidated. This study sought to identify key regulators that connect inflammatory FLS to chondrocyte dysfunction.</p> Methods <p>An integrated re-analysis of bulk and single-cell RNA sequencing data from OA synovial tissues was conducted to identify consistently upregulated genes. Functional assays, including migration and invasion tests, were performed on inflammatory FLS. FLS with silenced CYBB expression were co-cultured with chondrocytes to evaluate paracrine effects. small extracellular vesicle (sEV) isolation and tracking were utilized to assess intercellular communication. An OA rat model was employed for in vivo validation through local CYBB knockdown.</p> Results <p>CYBB expression was markedly upregulated in OA synovium, particularly within inflammatory FLS. Inhibition of CYBB reduced FLS inflammation, migration, and invasion. In co-culture experiments, inflammatory FLS induced chondrocyte inflammation and disrupted matrix gene expression in a manner dependent on CYBB. These effects were mediated by sEVs derived from fibroblast-like synoviocytes (FLS), with their inflammatory cargo being modified through CYBB silencing. In vivo experiments demonstrated that localized suppression of CYBB alleviated synovitis and cartilage degeneration.</p> Conclusions <p>CYBB serves as a pivotal mediator linking synovial inflammation to cartilage damage in osteoarthritis (OA), by mechanistically regulating the sEV-mediated communication from FLS to chondrocytes. Targeting CYBB could potentially represent a novel therapeutic strategy for OA.</p>