Hepatic conjugation plays important roles in systemic exposure and drug interactions of flavonoids. In the present study, the hepatic metabolism of calycosin, a major isoflavone from Astragali Radix, was characterized and the regioselectivity in the predominant glucuronidation pathway was first delineated in human liver microsomes (HLMs) and a panel of recombinant human UDP-glucuronosyltransferases (UGTs). Calycosin underwent major glucuronidation and minor oxidation and sulfation in human liver subcellular fractions. The major glucuronide (G2) of calycosin was isolated and identified as calycosin 3′-glucuronide by NMR analysis, and thus, the minor glucuronide (G1) was tentatively assigned as calycosin 7-glucuronide. The formations of both glucuronides in HLMs fit typical Michaelis-Menten kinetics. HLMs exhibited higher affinity (K, G2 12.37 ± 1.20 μM vs G1 40.90 ± 5.51 μM) and velocity (V, G2 5.39 ± 0.13 nmol/min/mg protein vs G1 2.80 ± 0.13 nmol/min/mg protein) on G2 formation, leading to the intrinsic clearance of calycosin via 3′-glucuronidation 6 times that through 7-glucuronidation. UGT1A1, 1A3 and 1A10 showed activities on both 3′-OH and 7-OH, whereas UGT1A7, 1A8, 1A9, and 2B7 were only capable of catalyzing 3′-OH glucuronidation of calycosin. Among them, UGT1A9 exhibited the highest activity (Cl, 2169.50 μL/min/mg protein) for 3′-glucuronide formation followed by UGT1A7 (Cl, 396.38 μL/min/mg protein). UGT1A1 showed the highest activity towards 7-OH glucuronidation (Cl, 224.34 μL/min/mg protein), which was comparable to its activity on 3′-OH glucuronidation (Cl, 203.82 μL/min/mg protein). Propofol (UGT1A9 inhibitor) produced a complete inhibition of 3′-glucuronide formation accompanied by an increase of 7-glucuronide in HLMs, while bilirubin (UGT1A1 inhibitor) only partially (∼60%) inhibited the 7-OH glucuronidation. These findings demonstrated the regioselective glucuronidation at the 3′-OH of the isoflavone calycosin in HLMs and shed light on potential drug interactions of calycosin with other UGT1A9 substrates. © 2014 Elsevier Ireland Ltd. All rights reserved.