Astragaloside IV (AIV) is the most abundant saponin and a marker compound in Astragali Radix, a Chinese herb notable for its anti-aging and immune-enhancing effects. The present study investigated the role of intestinal bacterial conversion in the in vivo fate of AIV administered through a traditional oral route for the first time. When incubated anaerobically with rat intestinal bacteria, AIV generated five metabolites with three [monoglycosides brachyoside B and cyclogaleginoside B, the aglycone cycloastragenol (CA)] via stepwise deglycosylation and two from further epimerization (CA-iso) and dehydrogenation (CA-2H). Hydrolytic removal of C-6 glucose was a rate-limiting step for formations of CA and its derivatives. When AIV was orally administered to the rat, CA and CA-iso presented as the main components in plasma following AIV, and the AUC(0-∞) were 88.60 ± 9.66 (CA), 179.06 ± 28.53 (CA-iso) and 452.28 ± 43.33 nM·h (AIV). CA-2H was the predominant form in feces but was not detected in urine or plasma. This agreed well with in vitro data including rapid hepatic metabolism of CA-2H to form CA and CA-iso and reversible conversions between CA-2H and CA/CA-iso by intestinal bacteria. These findings support a crucial role of gut bacterial conversion of AIV in the traditional application of Astragali herb and warrant further investigational emphasis on CA and CA-iso.