Flavonoids are polyphenolic compounds with various claimed health benefits, but the extensive metabolism by uridine-5'-diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in liver and intestine led to poor oral bioavailabilities. The effects of structural changes on the sulfonation of flavonoids have not been systemically determined, although relevant effects of structural changes on the glucuronidation of flavonoids had. We performed the regiospecific sulfonation of sixteen flavonoids from five different subclasses of flavonoids, which are represented by apigenin (flavone), genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol), and phloretin (chalcone). Additional studies were performed using 4 monohydroxyl flavonoids with a -OH group at the 3, 4', 5 or 7 position, followed by 5 dihydroxyl flavonoids, and 2 trihydroxyl flavonoids by using expressed human SULT1A3 and Caco-2 cell lysates. We found that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at the 7-OH position in Caco-2 cell lysates with minor amounts of 4'-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2 cell lysates were highly correlated at substrate concentrations of 2.5 and 10 μM. Molecular docking studies provided structural explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion, molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols, and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells.