Anthocyanidin synthase from Vitis vinifera (VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2R,3S,4S-cis-leucocyanidin, into 2R,4S-flavan-3,3,4-triol ([M + H]+, m/z 323) and quercetin. The C3-hydroxylation product 2R,4S-flavan-3,3,4-triol is first produced and its C3,C4-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C3-hydroxylation leading to a 4-keto-2R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2R,3S,4R-trans-leucocyanidin, is similarly transformed into quercetin upon C3,C4-dehydration, but unlike 3,4-cis-leucocyanidin, it also undergoes some C2,C3-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C4 to give traces of cyanidin. Overall, the C3,C4-trans isomer of leucocyanidin is transformed into 2R,4R-flavan-3,3,4-triol (M + 1, m/z 323), (+)-DHQ, (−)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4-cis-leucocyanidin- and 3,4-trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C3-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.