Here we sought to identify transcription factors that induce ADAMTS5, a crucial proteinase for osteoarthritis development. Exhaustive comparison of the genomic sequences of human, macaque, and mouse ADAMTS5 genes revealed that the proximal 1.4 kb of the 5'-end-flanking regions containing several consensus motifs was highly conserved. Among putative transcription factors for these motifs, NF-κB family member RelA/p65 most strongly stimulated the promoter activity. In the ADAMTS5 gene, there were three NF-κB binding motifs, in which deletion, mutagenesis, and tandem repeat analyses of the luciferase assay identified the core responsive elements of RelA/p65 to be -896/-887 and -424/-415 bp with specific bindings. The endogenous Adamts5 expression in ATDC5 cells was increased by RelA/p65 overexpression and decreased by knockdown through its siRNA. The expression was also inhibited by the Rela deletion through Cre transfection in primary articular chondrocytes from Rela(fl/fl) mice. In the ex vivo culture of femoral head cartilage from mesenchymal cell-specific Rela knock-out (Prx1-Cre;Rela(fl/fl)) mice, aggrecanolysis was significantly lower than that in the Rela(fl/fl) cartilage. Finally, in the experimental mouse osteoarthritis model, ADAMTS5 and RelA were co-localized in chondrocytes of degraded articular cartilage. We conclude that RelA/p65 is a potent transcriptional activator of ADAMTS5 in chondrocytes during osteoarthritis development.