Background Calycosin is one of the main bioactive and characteristic constituents in Radix Astragali (Chinese name Huangqi), a Chinese medicinal herb noted for its immune promotion function. The beneficial actions of calycosin have been evidenced by in vivo and in vitro studies. However, the in vivo fates and hence the bioactive forms of calycosin remain unknown. The present study characterized, for the first time, the in vivo forms of calycosin in the rat plasma and delineated its absorption and metabolic properties on in vitro models. Methods Calycosin (50 mg/kg) was orally administrated to male Sprague-Dawley rats and blood samples collected at appropriate time intervals. The intestinal permeability of calycosin was measured on a Caco-2 cell model. Hepatic metabolic stability of calycosin was examined in pooled human liver microsomes (HLMs) or S9 (HLS) fractions. Reaction phenotyping was conducted in HLMs and 12 recombinant UGTs. Structural identification was performed using HPLC-MS/MS and quantitative analysis completed using HPLC-UV. The major metabolite G2 was biosynthesized and its structure unambiguously identified by HPLC-MS/MS and NMR spectrometry. Results After oral administration, calycosin was absorbed rapidly then eliminated quickly from the rat plasma. Two metabolites of calycosin, 1 mono-sulfated and 1 mono-glucuronidated, were detected in the plasma. The mono-glucuronidated metabolite was the main drug-related component in the plasma. Bidirectional transport study on Caco-2 monolayer revealed a high permeability of calycosin (Papp values ~3×10 -5 cm/s in both absorptive and secretory directions). Calycosin underwent glucuronidation, sulfation and oxidation in human hepatic subcellular fractions with the average elimination rate of calycosin via glucuronidation >100 times those via other two pathways. Totally six metabolites, 2 mono-oxidated (O1, O2), 2 mono-glucuronidated (G1, G2) and 2 mono-sulfated (S1, S2) of calycosin, were tentatively identified. G2 was generated from a large-scale incubation, purified and unambiguously identified as calycosin-7-O-glucuronide. G2 was proved to be the glucuronidated metabolite observed in rat plasma. Kinetic studies showed that G2 formation by HLMs followed substrate inhibition kinetics (Km 13.58 uM; Vmax 5.65 nmol/min/mg protein). The recombinant human UGT1A9 exhibited the highest affinity to G2 (Km 4.39 uM; Vmax 9.11 nmol/min/mg protein). UGT1A7, 1A8, 1A1 and 1A3 also contributed but to a less extent. Propofol, the selective inhibitor of UGT1A9 could abolish G2 formation in a dose-dependent manner. Conclusions When taken orally, calycosin is absorbed rapidly, then undergoes extensive first-pass hepatic metabolism. Its glucuronidated metabolite formed in the liver is the main drug-related component in rat plasma. UGT 1A9 was the major isoform catalyzing calycosin glucuronidation in HLMs.