Tenascin-C (TN-C) is a key component of extracellular matrix (ECM) and its expression process is poorly understood during rheumatic heart valvular disease (RHVD). In this study, we found that interferon (IFN)-γ, tumour necrosis factor (TNF)-α and TN-C concentrations in patients with RHVD were significantly higher than in normal controls. More IFN-γ receptors and TNF receptors were found being expressed on rheumatic aortic valves interstitial cells than on non-rheumatic ones and their expression was patients' sera dependent. Antibodies neutralizing IFN-γ or TNF-α could attenuate patients' sera-induced TN-C transcription by isolated rheumatic aortic valves interstitial cells. By application with different protein kinase inhibitors, we found that combined with cyclic strain, TNF-α and IFN-γ induced TN-C transcription through the RhoA/ROCK signalling pathway. At the same time, p38 mitogen-activated protein kinase was involved in TNF-α and IFN-γ induced TN-C transcription. TNF-α also increased TN-C mRNA level by additional PKC and ERK 1/2 activation. Our finding revealed a new insight into ECM remodelling during RHVD pathogenesis and new mechanisms involved in the clinical anti-IFN-γ and anti-TNF-α therapy.