Xanthohumol (XN) is the principal prenylated flavonoid of the hop plant and has recently gained considerable interest due to its potential cancer-chemopreventive effects. However, the metabolism of XN has not yet been investigated in detail. Therefore, we studied the in vitro phase II metabolism of XN using nine human recombinant UDP-glucuronosyltransferases (UGT) and five sulfotransferases (SULT). The identification of the metabolites formed was elucidated using HPLC with diode array detection as well as HPLC/API-ES MS. XN was efficiently glucuronidated by UGT 1 A 8, 1 A 9, and 1 A 10; further important UGTs were UGT 1 A 1, 1 A 7, and 2 B 7. With respect to the sulfation reaction, SULT 1 A 1*2, 1 A 2, and 1 E 1 were the most active SULT forms. UGT 1 A 3, 1 A 4, and 1 A 6 as well as SULT 1 A 3 and 2 A 1 were of minor importance for the conjugation of XN. Three mono-glucuronides as well as three mono-sulfates were identified. Considering the tissue distribution of the tested UGT and SULT enzyme forms, these findings suggest a prominent role for the glucuronidation and sulfation of XN in the liver as well as in the gastrointestinal tract.