The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX−/−) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX−/− fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX−/− fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX−/− fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX−/− fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway.