Aim To observe the effect of TLR4/NF-κB signaling pathway on astragaloside IV inhibiting myocardial hypertrophy induced by isoproterenol (ISO). Methods The ISO ［5 mg/(kg·d)］ was used as myocardial hypertrophy models by intraperitoneal injection. Sixty SD rats were randomly assigned to the following six groups (10 rats for each group): normal group, ISO group, ISO plus astragaloside IV 20 mg/(kg·d) group, ISO plus astragaloside IV 40 mg/(kg·d) group, ISO plus astragaloside IV 80 mg/(kg·d) group, ISO plus propranolol 40 mg/(kg·d) group. Administered groups received continually intragastric administration for 3 weeks, and ISO were intraperitoneal injected as long as 2 weeks in the day after that. 3 weeks later, heart mass index (HMI) and left ventricular mass index (LVMI) of rats in each group were measured. HE staining was used for measuring transverse diameter of left ventricular myocardial cells (TDM). RT-PCR was used to quantify mRNA expression of ANP and TLR4, Western blot was used to quantify protein expression of TLR4, p65 and IκBα in the tissue, ELISA was used to quantify TNF-α and IL-6. Results Comparing the ISO group with the normal group, the differences were in the followings: the HMI and LVMI were significantly increased, the TDM were increased, the protein expression of TLR4 and p65 were increased, while the IκBα were decreased the expression of TLR4 and ANP mRNA were increased, TNF-α and IL-6 in serum were significantly increased. Comparing the other 3 groups (ISO plus astragaloside IV) with the ISO group, the differences were in the followings: the HMI and LVMI were significantly decreased, TDM were decreased, the protein expression of TLR4 and p65 were decreased, while the IκBα were increased;the expression of TLR4 and ANP mRNA were decreased, TNF-α and IL-6 in serum were significantly decreased, and the differences were positively related to dose of three groups. Conclusions astragaloside IV has a protective effect on cardiac hypertrophy induced by ISO, which is partially referred to inhibiting the TLR4/NF-κB signaling pathway and more than attenuating inflammatory effect.