The cGAS-STING pathway plays an important role in pathogen-induced activation of the innate immune response. 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) highly found in the synovial fluid of osteoarthritis (OA) patients increases the expression of catabolic factors via the toll-like receptor-2 (TLR-2) signaling pathway. In this study, we investigated whether 29-kDa FN-f induces inflammatory responses via the cyclic GMP-AMP synthase (cGAS)/STING pathway in human primary chondrocytes. The levels of cGAS and STING were elevated in OA cartilage compared to normal cartilage. Long-term treatment of chondrocytes with 29-kDa FN-f activated the cGAS/STING pathway together with increased level of gamma-H2AX, a marker of DNA breaks. In addition, expressions of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF-2), granulocyte colony-stimulating factor (G-CSF/CSF-3), and type I interferon (IFN), increased more than 100 folds in 29-kDa FN-f-treated chondrocytes. However, knockdown of cGAS and STING suppressed 29-kDa FN-f-induced expression of GM-CSF, G-CSF, and IFN- together with decreased activation of TBK1, IRF3, and IB. Furthermore, NOD2 or TLR-2 knockdown led to suppressed expression of GM-CSF, G-CSF, and IFN- as well as decreased activation of the cGAS/STING pathway in 29-kDa FN-f-treated chondrocytes. These data demonstrated that the cGAS/STING/TBK1/IRF3 pathway may play a critical role in 29-kDa FN-f-induced expression of pro-inflammatory cytokines.