The objective of this study was to determine the regiospecificity of the important uridine diphosphate glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of flavones and flavonols. We systematically studied the glucuronidation of 13 flavonoids (7 flavones and 6 flavonols, with hydroxyl groups at C-3, C-4', C-5, and/or C-7 positions in flavonoid structure) at a substrate concentration of 10 μM by 8 recombinant human UGT isoforms mainly responsible for the metabolism of flavonoids, UGTs 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, and 2B7. At 10 μM substrate concentration, different UGT isoforms gave different regiospecific glucuronidation patterns. UGT 1A1 equally glucuronidated 3-O (glucuronic acid substituted at C-3 hydroxyl group), 7-O, and 4'-O, whereas UGTs 1A8 and 1A9 preferably glucuronidated only 3-O and 7-O positions. UGT 1A1 usually showed no regiospecificity for glucuronidating any position, whereas UGT 1A8 and UGT 1A9 showed dominant, moderate, or weak regiospecificity for 3-O or 7-O position, depending on the structure of the compound. UGT 1A3 showed dominant regiospecificity for the 7-O position, whereas UGT 1A7 showed dominant regiospecificity for the 3-O position. We also showed that the glucuronidation rates of 3-O and 7-O positions in flavones and flavonols were affected by the addition of multiple hydroxyl groups at different positions as well as by the substrate concentrations (2.5, 10, and 35 μM). In conclusion, regiospecific glucuronidation of flavonols was isoform- and concentration-dependent, whereas flavones were dominantly glucuronidated at the 7-O position by most UGT isoforms. We also concluded that UGTs 1A3 and 1A7 showed dominant regiospecificity for only the 7-O and 3-O positions, respectively. UGTs 1A8 and 1A9 showed moderate or weak preference on glucuronidating position 3-O over the 7-O position, whereas other UGT isoforms did not prefer glucuronidating any particular positions.