Fibronectin fragments found in synovial fluid of patients with osteoarthritis (OA) induce the catabolic responses in cartilage. Nuclear high mobility group protein Box 1 (HMGB1), a damage-associated molecular pattern, is responsible for the regulation of signaling pathways related with cell death and survival in response to various stimuli. In this study, we investigated whether 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)-induced change in HMGB1 expression influences the pathogenesis of OA through HMGB1-modulated autophagy signaling pathway. Human articular chondrocytes were enzymatically isolated from articular cartilage. The level of mRNA was measured by quantitative real-time PCR. The expression of proteins was examined by western blot analysis, immnunofluorescence assay, and enzyme-linked immunosorbent assay. Interaction of proteins was evaluated by immunoprecipitation. The HMGB1 level was significantly reduced in human OA cartilage compared to normal cartilage. Although 29-kDa FN-f significantly reduced the HMGB1 expression at the mRNA and protein levels 6 h after treatment, the cytoplasmic level of HMGB1 was increased in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f significantly inhibited the interaction of HMGB1 with Beclin-1 but increased the interaction of Bcl-2 with Beclin-1, together with decreased levels of Beclin-1 and phosphorylated Bcl-2. In addition, the level of microtubule associated protein 1 light chain 3-II, an autophagy marker, was down-regulated in 29-kDa FN-f-treated chondrocytes, whereas the effect was antagonized by mTOR knockdown. Furthermore, prolonged treatment with 29-kDa FN-f significantly increased the release of HMGB1 into the culture medium. These results demonstrated that 29-kDa FN-f inhibits chondrocyte autophagy through modulation of HMGB1 signaling pathway.