Nucleotide-binding and oligomerization domain (NOD) is innate pattern recognition receptor that recognizes pathogen-associated molecular patterns and damage-associated molecular patterns. 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 is involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR), and western blot analysis. Small interfering RNA was used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). Immunoprecipitation assay was performed to examine interaction of proteins. NOD2 level in human OA cartilage was much higher than in normal cartilage. NOD1 and NOD2 expressions were upregulated by 29-kDa FN-f in human chondrocytes but also pro-inflammatory cytokines, including interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). NOD2 silencing showed that NOD2 was involved in 29-kDa FN-f-induced expressions of TLR-2 as well as IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13, which were also suppressed by TLR-2 knockdown. Furthermore, NOD2 or TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors receptor-interacting protein 2 (RIP2) and myeloid differentiation 88 in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.