Astragaloside IV, a natural product purified from the Chinese medical herb Astragalus membranaceus (Fisch) Bge, is now being developed as a cardioprotective agent for treating cardiovascular diseases. The purpose of the present study was to examine in vivo pharmacokinetics and tissue distribution in both rats and dogs by using an established high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry quantitative detection method. Astragaloside IV showed moderate to fast elimination; the elimination half-life of astragaloside IV was 98.1, 67.2 and 71.8 min in male rats, and 34.0, 66.9 and 131.6 min in female rats at doses of 0.75, 1.5 and 3.0 mg/kg, respectively. There was no significant difference in systemic clearance at three dose levels, suggesting that astragaloside IV may have linear pharmacokinetic characteristics in rats within the dose ranges tested. The highest concentration of astragaloside IV was detected in the lung and liver. However, limited distribution to the brain, indicates that astragaloside IV may have difficulty penetrating the blood brain barrier. In addition, only about 50% of the parent astragaloside IV was recovered in both urine and feces. These results indicate that there was about 83% astragaloside IV binding to plasma protein and that the binding to the plasma is linear at the concentration range of 250-1000 ng/ml. As in rats, astragaloside IV may have linear pharmacokinetic characteristics in dogs within the dose ranges tested. Astragaloside IV was slowly cleared via hepatic clearance with a systemic clearance (CL) of about 0.004 l/kg/min. Based on the favorable pharmacokinetic properties in both rats and dogs, astragaloside IV warrants further investigation for the prevention of cardiovascular diseases.