Aim To investigate the effects of RNA interference （RNAi） downregulating TLR4 of mice with isoproterenol （ISO） induced myocardial ischemic necrosis. Methods A myocardial ischemic necrosis model of mice was established by abdominal cavity injection of ISO for 3 days. The rats were divided into four groups: control group, myocardial ischemic group （MI group） and myocardial ischemic treated with negative plasmid group （PCN group）, myocardial ischemic treated with siRNA-TLR4 plasmid group （siRNA-TLR4 group）. Inhibition of TLR4 by siRNA-TLR4 plasmid was constructed. Pathological changes of myocardial ischemic were observed through myocardial tissue stained with HE. The expression level of TLR4 was detected by Western blot. The variation of cardiomyocytes apoptosis was measured with TUNEL.Results The expressions of protein of TLR4 in siRNA-TLR4 group were significantly decreased compared with those of control group, PCN group, and MI group. Compared to MI group and PCN group, myocardial ischemic necrosis was relieved in the siRNA-TLR4 group. And numbers of apoptosis cardiomyocytes in siRNA-TLR4 group were lower than those in MI group and PCN group （9.39%±2.61% vs. 61.44%±2.34% and 57.21%±4.40%）. Conclusions RNAi can inhibit TLR4 expression and ameliorat myocardial ischemic necrosis induced by ISO efficiently. It may be promising as a new treatment of acute myocardial ischemic necrosis.