Ginsenosides are a group of tetracyclic triterpenoids with promising health benefits, consisting of ginseng aglycone attached to various glycans. Pq3-O-UGT2, an important UDP-dependent glycosyltransferase (UGT), catalyzes the production of Ginsenoside Rg3 and Rd by extending the glycan chain of Ginsenoside Rh2 and F2, respectively, with higher selectivity for F2. However, the mechanism underlying its substrate recognition remains unclear. In this study, the crystal structures of Pq3-O-UGT2 in complex with its acceptor substrates are solved. The structures revealed a Nα5-oriented acceptor binding pocket in Pq3-O-UGT2, shaped by the unique conformation of the Nα5-Nα6 linker. Hydrophobic interactions play a pivotal role in the recognition of both Rh2 and F2, while hydrogen bonds specifically aid in F2 recognition due to its additional glucose moiety. The hydrophobic nature of the acceptor binding pocket also enables Pq3-O-UGT2 to recognize flavonoids. Overall, this study provides novel insights into the substrate recognition mechanisms of ginsenoside UGTs, advancing the understanding of their function and specificity.