Astragaloside is a monomer isolated from Astragalus membranaceus, a flowering plant in the family Fabaceae. The aim of the present study was to investigate the anti-apoptotic affect of astragaloside on myocardial cells through the TLR4/NF-kappa B signaling pathway. Astragaloside, NF-kappa B inhibitor pyrrolidine dithiocarbamate (PDTC) and Toll-like receptor 4 (TLR4) blocking antibody solution were prepared in vitro, and myocardial cells were incubated and cultured in serum-free medium overnight. Cells were divided into five groups: the normal control group, serum-free group, astragaloside group, TLR4 blocking antibody group and NF-kappa B inhibitor PDTC group. The myocardial cell apoptosis in each group was detected using flow cytometry, and the expression levels of TLR4 and NF-kappa B were detected via western blotting. The apoptosis rate in the serum-free group was significantly higher than that in the normal control group. The apoptosis rate of myocardial cells in the TLR4 blocking antibody group and NF-kappa B inhibitor PDTC group was lower than that in the serum-free group. In addition, the myocardial cell apoptosis was more obviously decreased in the astragaloside group, and the protein expression levels of TLR4 and NF-kappa B in the serum-free group were significantly higher than those in normal control group. The protein expression levels of TLR4 and NF-kappa B in the astragaloside group were obviously lower than those in the serum-free group, and the protein expression levels of TLR4 and NF-kappa B in the TLR4 blocking antibody group and NF-kappa B inhibitor PDTC group were decreased. In conclusion, astragaloside reduced myocardial cell apoptosis and protected myocardial cells, which may be one of the mechanisms of a traditional Chinese medicine monomer in treating heart failure.