Background: Myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide, and its main pathophysiological mechanism is myocardial ischemia/reperfusion (I/R) injury. TNC is an extracellular matrix glycoprotein, and high TNC expression was associated with MI and ventricular remodeling. The present study aimed to investigate the effect and the underlying mechanisms of TNC on myocardial I/R injury.Methods: Cardiomyocyte H9c2 cells was used to establish an in vitro hypoxia/reoxygenation (H/R) model, which were under hypoxia for 4 h and reoxygenation for 12 h. TNC was silenced by small interfering RNA (siRNA) in H9c2 cells.Results: TNC mRNA and protein expressions were increased by H/R. TNC knockdown by siRNA improved the cell viability in H/R-stimulated H9c2 cells. TNC knockdown reversed the H/R-induced increase in the apoptosis, as evidenced by reduced TUNEL+ cells and caspase-3 activity, and inhibited oxidative stress and inflammatory cytokine production. TNC silencing inhibits TLR4 mRNA and protein expressions and NF-kappa B signals, as evidenced by reduced nuclear NF-κB p65 and increased cytoplasmic I-κBα in H/R-stimulated H9c2 cells. All these cardioprotection against H/R by siTNC was reversed by TLR4 overexpression.Conclusions: TNC silencing prevents H/R-induced injury by inhibiting apoptosis and oxidative stress by inhibition of TLR4-NF-κB pathway.